Methods and compositions relating to improved lentiviral vector production systems

ABSTRACT

The present invention provides HIV-derived lentivectors which are multiply modified to create highly safe, efficient, and potent vectors for expressing transgenes for gene therapy. The lentiviral vectors comprise various combinations of an inactive central polypurine tract, a stuffer sequence, which may encode drug susceptibility genes, and a mutated hairpin in the 5′ leader sequence that substantially abolishes replication. These elements are provided in conjunction with other features of lentiviral vectors, such as a self-inactivating configuration for biosaftey and promoters such as the EF1α promoter as one example. Additional promoters are also described. The vectors can also comprise additional transcription enhancing elements such as the wood chuck hepatitis virus post-transcriptional regulatory element. These vectors therefore provide useful tools for genetic treatments for inherited and acquired disorders, gene-therapies for cancers and other disease, the creation of industrial and experimental production systems utilizing transformed cells, as well as for the study of basic cellular and genetic processes.

The present application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/309,569 filed Aug. 2, 2001, the entire text ofwhich is herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to improved lentiviral vectors, theirproduction and their safe use in gene delivery and expression of desiredtransgenes in target cells.

2. Description of Related Art

Transfection of cells is an increasingly important method of deliveringgene therapy and nucleic acid based treatment for a number of disorders.Transfection is the introduction of nucleic acids into recipienteukaryotic cells and the subsequent integration of the nucleic acidsequence into chromosomal DNA. Efficient transfection requires vectors,which facilitate the introduction of foreign nucleic acids into thedesired cells, may provide mechanisms for chromosomal integration, andprovide for the appropriate expression of the traits or proteins encodedby those nucleic acids. The design and construction of efficient,reliable, and safe vectors for cell transfection continues to be asubstantial challenge to gene therapy and treatment methods.

Viruses of many types have formed the basis for vectors. Virus infectioninvolves the introduction of the viral genome into the host cell. Thatproperty is co-opted for use as a gene delivery vehicle in viral basedvectors. The viruses used are often derived from pathogenic viralspecies that already have many of the necessary traits and abilities totransfect cells. However, not all viruses will successfully transfectall cell types at all stages of the cell cycle. Thus, in the developmentof viral vectors, viral genomes are often modified to enhance theirutility and effectiveness for introducing foreign gene constructs(transgenes) or other nucleic acids. At the same time, modifications maybe introduced that reduce or eliminate their ability to cause disease.

Lentiviruses are a subgroup of retroviruses that can infect nondividingcells owing to the karyophilic properties of their preintegrationcomplex, which allow for its active import through the nucleopore.Correspondingly, lentiviral vectors derived from human immunodeficiencyvirus type 1 (HIV-1) can mediate the efficient delivery, integration andlong-term expression of transgenes into non-mitotic cells both in vitroand in vivo (Naldini et al., 1996a; Naldini et al., 1996b; Blomer etal., 1997). For example, HIV-based vectors can efficiently transducehuman CD34⁺ hematopoietic cells in the absence of cytokine stimulation(Akkina et al., 1996; Sutton et al., 1998; Uchida et al., 1998; Miyoshiet al., 1999; Case et al., 1999), and these cells are capable oflong-term engraftment in NOD/SCID mice (Miyoshi et al., 1999).Furthermore, bone marrow from these primary recipients can repopulatesecondary mice with transduced cells, confirming thelentivector-mediated genetic modification of very primitivehematopoietic precursors, most probably bona fide stem cells. Since noneof the other currently available gene delivery systems has such anability, lentiviral vectors provide a previously unexplored basis forthe study of hematopoiesis and similar phenomena, and for the genetherapy of inherited and acquired disorders via the genetic modificationof human stem cells (HCLs).

This important capability is subject to significant biosafety concerns(Akkina et al., 1996; Sutton et al., 1998; Uchida et al., 1998). Theaccidental generation of replication-competent recombinants (RCRs)during the production of lentiviral vector stocks represents one of themajor problems to be solved before lentiviral vectors can be consideredfor human gene therapy.

In the retroviral genome, a single RNA molecule that also contains allthe necessary cis-acting elements carries all the coding sequences.Biosafety of a vector production system is therefore best achieved bydistributing the sequences encoding its various components over as manyindependent units as possible, to maximize the number of crossovers thatwould be required to re-create an RCR. Lentivector particles aregenerated by co-expressing the virion packaging elements and the vectorgenome in host producer cells, e.g. 293 human embryonic kidney cells. Inthe case of HIV-1-based vectors, the core and enzymatic components ofthe virion come from HIV-1, while the envelope protein is derived from aheterologous virus, most often VSV due to the high stability and broadtropism of its G protein. The genomic complexity of HIV, where a wholeset of genes encodes virulence factors essential for pathogenesis butdispensable for transferring the virus genetic cargo, substantially aidsthe development of clinically acceptable vector systems.

Multiply attentuated packaging systems typically now comprise only threeof the nine genes of HIV-1: gag, encoding the virion main structuralproteins, pol, responsible for the retrovirus-specific enzymes, and rev,which encodes a post-transcriptional regulator necessary for efficientgag and pol expression (Dull, et al., 1998). From such an extensivelydeleted packaging system, the parental virus cannot be reconstituted,since some 60% of its genome has been completely eliminated. In oneversion of an HIV-based packaging system, Gag/Pol, Rev, VSV G and thevector are produced from four separate DNA units. Also, the overlapbetween vector and helper sequences has been reduced to a few tens ofnucleotides so that opportunities for homologous recombination areminimized.

HIV type 1 (HIV-1) based vector particles may be generated byco-expressing the virion packaging elements and the vector genome in aso-called producer cell, e.g. 293T human embryonic kidney cells. Thesecells may be transiently transfected with a number of plasmids.Typically from three to four plasmids are employed, but the number maybe greater depending upon the degree to which the lentiviral componentsare broken up into separate units. Generally, one plasmid encodes thecore and enzymatic components of the virion, derived from HIV-1. Thisplasmid is termed the packaging plasmid. Another plasmid encodes theenvelope protein(s), most commonly the G protein of vesicular stomatitisvirus (VSV G) because of its high stability and broad tropism. Thisplasmid may be termed the envelope expression plasmid. Yet anotherplasmid encodes the genome to be transferred to the target cell, thatis, the vector itself, and is called the transfer vector. Recombinantviruses with titers of several millions of transducing units permilliliter (TU/ml) can be generated by this technique and variantsthereof. After ultracentrifugation concentrated stocks of approximately10⁹ TU/ml can be obtained.

The vector itself is the only genetic material transferred to the targetcells. It typically comprises the transgene cassette flanked bycis-acting elements necessary for its encapsidation, reversetranscription, nuclear import and integration. As has been previouslydone with oncoretroviral vectors, lentiviral vectors have been made thatare “self-inactivating” in that they lose the transcriptional capacityof the viral long terminal repeat (LTR) once transferred to target cells(Zufferey, et al. 1998). This modification further reduces the risk ofemergence of replication competent recombinants (RCR) and avoidsproblems linked to promoter interference.

Nevertheless, experience with retroviral vectors demonstrates that theemergence of a replication-competent retrovirus (RCR) is possible,although a rare event even when vectors are produced by stable packagingcell lines and components designed to provide high safety. Thepathogenic potential of RCRs is demonstrated by the induction of cancerin monkeys injected with contaminated oncoretroviral vector stocks.Consequently, the administration of retroviral vectors to human patientsis authorized only if the presence of contaminant RCRs has been excludedby a test sensitive enough to detect a single RCR in an aliquot equal to5% of the dose actually used. Creating highly safe vectors is clearlyimportant when doses equal or superior to 10¹⁰ transducing units may benecessary to reach therapeutic efficiency.

There is therefore a significant need to develop improved lentivirusesfor use as transducing vectors capable of effectively transducing cellsand expressing desired transgenes at high levels while meeting biosafetyrequirements. Currently available lentiviral vector production systemsrely on the expression of packaging and vector elements either bytransient transfection or in stable cell lines. Deletion ofnon-essential genes from the parental virus and splitting of the vectorsystem components on separate DNA units act to help minimize the risk ofemergence of RCRs. Greatest safety is achieved with the fewest, or,ideally, with zero RCR occurrence in vector production. The presentinvention utilizes specific changes in the packaging and vector systemcomponents, their methods of production and their methods of use inorder to further reduce or eliminate the occurrence of RCR.

SUMMARY OF THE INVENTION

The present invention provides for compositions and methods that improvethe biosafety of lentiviral vector production systems in such a waythat, if its components undergo multiple recombination eventsreconstituting the parental virus, the resulting recombinant will stillbe defective with respect to the ability to proceed through subsequentinfection and replication. The invention further improves the biosafetyof lentiviral vector production by optionally providing for drugsensitivity for any resulting recombinants.

The present invention thus concerns, in a general and overall sense,improved vectors and methods for the production thereof that aredesigned to permit the safe transfection and transduction of animalcells, particularly human cells, and more particularly hematopoieticprogenitor cells, or stem cells (hHSC). The present inventionfacilitates appropriate expression of desired transgenes in such cellsby providing effective vectors with increased safety.

The viral vectors of the present invention, therefore, may be generallydescribed as recombinant vectors that include at least the lentiviralgag and pol genes, that is, those genes required for virus production,which permit their manufacture in reasonable quantities using availableproducer cell lines. To meet important human safety needs, the morepreferred vectors in accordance with the present invention will notinclude any other active lentiviral genes, such as vpr, vif, vpu, nef,tat, such as where these genes have been removed or otherwiseinactivated. In fact, it is preferred that the only active lentiviralgenes present in the vector will be at most the aforementioned gag andpol genes, supplemented by the rev gene as may be required for efficientcyctoplasmic export and expression of vector genes.

The most preferred lentiviral genes and cis-acting sequence elements(e.g., long terminal repeats or LTRs, the psi signal, the RRE) used inpreparing lentivectors in accordance with the present invention will beone that is human immunodeficiency virus (HIV) derived, and moreparticularly, HIV-1 derived. Thus, the gag, pol and rev genes willpreferably be HIV genes and more preferably HIV-1 genes. However, thegag, pol and rev genes and cis-acting sequence elements from otherlentiviruses may be employed for certain applications in accordance withthe present invention, including the genes and cis-acting sequenceelements of HIV-2, simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FIV), bovine immunodeficiency virus (BIV),Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV),caprine arthritis encephalitis virus (CAEV) and the like. Suchconstructs could be useful, for example, where one desires to modifycertain cells of non-human origin. However, the HIV based vectorbackbones (i.e., HW cis-acting sequence elements and HIV gag, pol andrev genes) will generally be preferred in connection with most aspectsof the present invention in that HIV-based constructs are the mostefficient at transduction of human cells.

The most preferred configuration of the packaging elements is one inwhich the gag, pol and rev genes are present. However, the need for revmay be alleviated in some designs by using cis-acting sequencesfacilitating the cytoplasmic export of incompletely spliced RNAs in theabsence of Rev, the so-called constitutive RNA export element or CTEsuch as found in Mason-Pfizer monkey virus (Bray et al., 1994).Alternatively, specific codons may be altered in the gag and pol genesto similar effect (Kotsopoulou, et al., 2000). Also, components of thepol gene such as the integrase can be provided in a trans configuration,for instance as a VPR-integrase fusion protein (Wu, et al., 2000).

In the lentivectors of the present invention it is particularlydesirable to employ mutations in the central polypurine tract (cPPT) ofthe sequence encoding the lentiviral Gag/Pol polyprotein in thepackaging plasmid such that the introduced mutations interfere withlentiviral replication relative to wild-type genome. Such constructsprovide a biosafety feature in that the nuclear import ofreplication-competent recombinants. This feature greatly minimizes therisk that (RCRs) will emerge. The cPPT/cTS region need be inactive onlyon the packaging plasmid construct to confer this safety feature.Indeed, an active copy of the cPPT/cTS region is typically provided onthe transfer vector plasmid.

It is also desirable to employ in the present invention an additionalsequence element in the packaging plasmid encoding the lentiviralGag/Pol polyprotein in order to increase the genome length of anypotential recombinant lentiviruses such that the effects of mutation inthe central polypurine tract are maximized. This feature also minimizesthe risk of producing RCRs. The long sequence element may be introducedinto the vector genome at various positions that provide for maximizingthe effects of the mutations in the central polypurine tract of thesequence encoding the lentiviral Gag/Pol polyprotein. A particularlypreferred position is between the end of the pol or gag genes and thebeginning of the RRE sequence element.

In another preferred aspect of the invention, such long sequenceelements encode one or more genes conferring susceptibility to drugscurrently used with success to treat viral infection. One such sequenceelement may include sequence that encodes a thymidinekinase, or theIRES-tk cassette. One skilled in the art will recognize, of course, thatany such drug susceptibility gene or genes, or any like construct may beemployed to similar effect.

In a further preferred aspect of the invention, the 5′ LTR R-U5 regionof the vector plasmid contains a set of mutations that additionallyprevent the replication of putative viral recombinants. Such mutationspreferably include changes that either destabilize or excessivelystabilize the Poly(A) hairpin motif, which leads to reduced replicationof any RCRs.

One of skill in the art will recognize that the ultimate efficacy ofthese various aspects of the invention will depend upon the particularcombination of aspects employed. It is preferred that the mutantsequences of the Poly(A) hairpin structures in the 5′ LTR R-U5 region ofthe vector plasmid are to be used in conjunction with other preferredaspects. It is also contemplated that the invention may be embodied asvarious combinations of the individually described embodiments,including a combination of all disclosed embodiments, only two of thedisclosed embodiments, or, employed singly in the making and using ofsuch lentiviral vectors in the transfection and transduction of cells.In a most preferred embodiment of the present invention all theseaspects of the present invention will be present.

The present invention describes gene transfer vehicles that appearparticularly well suited for the transduction of cells and for theexpression of transgenes in various cell types. These compositions andmethods will facilitate the safe use of lentiviral vectors for thegenetic manipulation of cells, and should be particularly useful forboth research and therapeutic applications.

It will be understood by the skilled artisan that the invention is notlimited to any one particular cell type and that one may use thelentiviral vectors and methods of the invention for the expression oftransgenes in many cell types. Some examples of cell types contemplatedinclude terminally differentiated cells such as neurons, lung cells,muscle cells, liver cells, pancreatic cells, endothelial cells, cardiaccells, skin cells, bone marrow stromal cells, ear and eye cells.Additionally, stem cells and progenitor cells such as pancreatic ductalcells, neural precursors, and mesodermal stem cells are alsocontemplated. Most notably, however, the more preferred lentivectors ofthe present invention have highly desirable features that permit thehigh level expression of transgenes in human progenitor cells whilemeeting human biosafety requirements.

It is believed that the lentivectors of the present invention may beemployed to deliver any transgene that one desires, depending on theapplication. In the case of delivery to hematopoietic progenitor cells,one will typically select a transgene that will confer a desirablefunction on such cells, including, for example, globin genes,hematopoietic growth factors, which include erythropoietin (EPO), theinterleukins (such as Interleukin-1 (IL-1), Interleukin-2 (IL-2),Interleukin-3 (IL-3), Interleukin-6 (IL-6), Interleukin-12 (IL-12),etc.) and the colony-stimulating factors (such as granulocytecolony-stimulating factor, granulocyte/macrophage colony-stimulatingfactor, or stem-cell colony-stimulating factor), the platelet-specificintegrin αIIbβ, multidrug resistance genes, the gp91 or gp 47 genes thatare defective in patients with chronic granulomatous disease (CGD),antiviral genes rendering cells resistant to infections with pathogenssuch as human immunodeficiency virus, genes coding for blood coagulationfactors VIII or IX which are mutated in hemophiliacs, ligands involvedin T cell-mediated immune responses such as T cell antigen receptors, Bcell antigen receptors (immunoglobulins), the interleukin receptorcommon γ chain, as well as combination of T and B cell antigen receptorsalone or in combination with single chain antibodies such as ScFv, tumornecrosis factor (TNF), IL-2, IL-12, gamma interferon, CTLA4, B7 and thelike, genes expressed in tumor cells such as Melana, MAGE genes (such asMAGE-1, MAGE-3), P198, P1A, gp100 etc.

A principal application of the present invention will be to provide forvectors that deliver desired transgenes to hematopoietic cells for anumber of possible reasons. This might include, but of course not belimited to, the treatment of myelosupression and neutropenias which maybe caused as a result of chemotherapy or immunosupressive therapy orinfections such as AIDS, genetic disorders, cancers and the like.

Exemplary genetic disorders of hematopoietic cells that are contemplatedinclude sickle cell anemia, thalassemias, hemaglobinopathies, Glanzmannthrombasthenia, lysosomal storage disorders (such as Fabry disease,Gaucher disease, Niemann-Pick disease, and Wiskott-Aldrich syndrome),severe combined immunodeficiency syndromes (SCID), as well as diseasesresulting from the lack of systemic production of a secreted protein,for example, coagulation factor VIII and/or IX. In such cases, one woulddesire to introduce transgenes such as globin genes, hematopoieticgrowth factors, which include erythropoietin (EPO), the interleukins(especially Interleukin-1, Interleukin-2, Interleukin-3, Interleukin-6,Interleukin-12, etc.) and the colony-stimulating factors (such asgranulocyte colony-stimulating factor, granulocyte/macrophagecolony-stimulating factor, or stem-cell colony-stimulating factor), theplatelet-specific integrin αIIbβ, multidrug resistance genes, the gp91or gp 47 genes which are defective in patients with chronicgranulomatous disease (CGD), antiviral genes rendering cells resistantto infections with pathogens such as human immunodeficiency virus, genescoding for blood coagulation factors VIII or IX which are mutated inhemophiliacs, ligands involved in T cell-mediated immune responses suchas T cell antigen receptors, B cell antigen receptors (immunoglobulins),the interleukin receptor common γ chain, a combination of both T and Bcell antigen receptors alone and/or in combination with single chainantibodies (ScFv), IL2, IL12, TNF, gamma interferon, CTLA4, B7 and thelike, genes expressed in tumor cells such as Melana, MAGE genes (such asMAGE-1, MAGE-3), P198, P1A, gp100 etc.

Exemplary cancers are those of hematopoietic origin, for example,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof. Exemplary myeloid disorders include, but are not limited to,acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) andchronic myelogenous leukemia (CML). Lymphoid malignancies which may betreated utilizing the lentivectors of the present invention include, butare not limited to acute lymphoblastic leukemia (ALL) which includesB-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas contemplated as candidates for treatment utilizing thelentiviral vectors of the present invention include, but are not limitedto non-Hodgkin lymphoma and variants thereof, peripheral T-celllymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T-celllymphoma (CTCL), large granular lymphocytic leukemia (LGF) and Hodgkin'sdisease.

In other embodiments, the present invention is directed to host cellsthat have been transduced with one of the foregoing lentivectors. It isbelieved that the lentivectors of the present invention can be employedto transduce most any cell. Exemplary cells include but are not limitedto a CD4⁺T cell, a peripheral blood lymphocyte cell, a peripheral bloodmononuclear cell, a hematopoietic stem cell, a fetal cord blood cell, afibroblast cell, a brain cell, a lung cell, a liver cell, a muscle cell,a pancreatic cell, an endothelial cell, a cardiac cell, a skin cell, abone marrow stromal cell, and an eye cells, a pancreatic ductal cell, aneural precursor, a mesodermal stem cell and the like. The cellstransduced may further be primate, murine, porcine, or human in origin,or come from another animal species.

For the production of virus particles, one may employ any cell that iscompatible with the expression of lentiviral Gag and Pol genes, or anycell that can be engineered to support such expression. For example,producer cells such as 293T cells, TE 671 and HT1080 cells may be used.

Of course, as noted above, the lentivectors of the invention will beparticularly useful in the transduction of human hematopoieticprogenitor cell or a hematopoietic stem cell, obtained either from thebone marrow, the peripheral blood or the umbilical cord blood, as wellas in the tranduction of a CD4⁺T cell, a peripheral blood B or Tlymphocyte cell, a peripheral blood mononuclear cell, a dendritic cell,and a monocytic cell. Particularly preferred targets are CD34⁺ cells.

In still other embodiments, the present invention is directed to amethod for transducing a human hematopoietic stem cell comprisingcontacting a population of human cells that include hematopoietic stemcells with one of the foregoing lentivectors under conditions to effectthe transduction of a human hematopoietic progenitor cell in saidpopulation by the vector. The stem cells may be transduced in vivo or invitro, depending on the ultimate application. Even in the context ofhuman gene therapy, such as gene therapy of human stem cells, one maytransduce the stem cell in vivo or, alternatively, transduce in vitrofollowed by infusion of the transduced stem cell into a human subject.In one aspect of this embodiment, the human stem cell can be removedfrom a human, e.g., a human patient, using methods well known to thoseof skill in the art and transduced as noted above. The transduced stemcells are then reintroduced into the same or a different human.

Where a human subject is treated directly by introduction of the vectorinto the subject, the treatment is typically carried out by intravenousadministration of the vector. When cells, for instance CD34⁺ cells,dendritic cells, peripheral blood cells or tumor cells are transduced exvivo, the vector particles are incubated with the cells using a dosegenerally in the order of between 1 to 50 multiplicities of infection(MOI) which also corresponds to 1×10⁵ to 50×10⁵ transducing units of theviral vector per 10⁵ cells. This of course includes amount of vectorcorresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, and 50 MOI. Typically, the amount of vector may be expressed interms of HeLa transducing units (TU). Other routes for vectoradministration include intrarterially, endoscopically, intralesionally,percutaneously, subcutaneously, intramuscular, intrathecally,intraorbitally, intradermally, intraperitoneally, transtracheally,subcuticularly, by intrasternal injection, by inhalation or intranasalspraying, by endotracheal route and the like. In embodiments concerningtumor/cancer therapies with the vectors of the invention the expressionvector can be delivered by direct injection into the tumor or into thetumor vasculature.

A typical example of ex vivo gene therapy is a patient suffering fromchronic granulatous disease (CGD), whose CD34⁺ cells can be isolatedfrom the bone marrow or the peripheral blood and transduced ex vivo witha lentivector expressing the gp91phox gene before reimplantation. In thecase of patients suffering from severe combined immunodeficiency (SCID),the inventors contemplate a similar approach, using lentivectors of theinvention expressing the gene defective in the patient, for example, thegene encoding the common gamma chain of the Interleukin receptor. Forthe genetic treatment of HIV infection, the present inventorscontemplate intracellular immunization, wherein cells are renderedresistant to the HIV virus through the introduction of antiviral genes.In embodiments of the intracellular immunization for HIV, targets of thelentivectors of the invention include hematopoietic progenitors,peripheral blood CD4⁺ T cells, and monocytes. As will be recognized bythe skilled artisan, similar intracellular immunization methods can beused for other viral infections as well. For the immunotherapy ofcancers, tumor cells or antigen presenting cells such as dendritic cellswill be genetically engineered with the lentivectors of the invention.For cancer therapies some transgenes that may be used in the lentivectorconstructs of the invention are those that can inhibit, and/or kill,and/or prevent the proliferation, and/or mediate the apoptosis of, thecancer/tumor cell and/or genes such as TNF.

The lentivectors described herein may also be used in vivo, by directinjection into the blood or into a specific organ. For example, in oneembodiment intracerebral injection of lentivectors expressing the GlialCell Derived Nerve Growth Factor (GDNF), can be used for the treatmentof Parkinson's disease. In another example, intraportal injection of alentivector expressing coagulation factor VIII for the correction ofhemophilia A is envisioned. In yet another example, intravenous orintramuscular injection of a lentivector of the present inventionexpressing the dystrophin gene for the treatment of Duchenne MuscularDystrophy is envisioned. Thus, one of ordinary skill in the art willappreciate the extensive use of the lentivector constructs of thepresent invention in terms of gene therapies.

As used herein the specification or claim(s) when used in conjunctionwith the word “comprising”, the words “a” or “an” may mean one or morethan one. As used herein “another” may mean at least a second or more.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Schematic drawing of pMDLg/pRRE and pMDLD. The cPPT/cTS sequenceelement (black box) is indicated on the pol gene of pMDLg/pRRE. Theplasmid pMDLD is a modified version with multiple mutations in the cPTTsequence element abolishing its function. Sequence comparison betweenthe two plasmids is shown at the bottom.

FIG. 2. Inactivation of the cPPT sequence element in the packagingsystem does not affect vector production. Vectors transducing GFP wereproduced in parallel with packaging systems having or lacking afunctional cPPT sequence element by transient transfection of 293Tcells. Vector stocks were matched for their reverse transcriptaseactivity and used to transduce 293T cells. Two days later, thepercentage of GFP positive cells was determined by FACS. Vector titerswere identical whether the packaging system had a functional or amutated cPPT.

FIG. 3. Strategies to increase the length of packaging plasmids. Tomaximize the benefit of the cPPT inactivation, genome length ofrecombinant lentiviruses must be as long as possible. To this end DNAsequence can be inserted between the pol gene and the RRE sequenceelement. The inserted DNA can work either as a stuffer only or may bechosen to fulfill additional functions. One possibility is the use of agene conferring drug sensitivity to cells infected by the recombinantlentiviruses e.g. the thymidinekinase gene (tk) from the Herpes simplexvirus (HSV). An internal ribosomal entry site (IRES) is placed upstreamof the tk gene to allow its efficient expression.

FIG. 4. Mutations known for their strong inhibitory effect on HIV-1replication were introduced in the R-U5 region of HIV-1 based vectorstransducing the GFP gene.

FIG. 5. Mutations in the R-U5 region of lentivirus vectors do notcompromise their transduction efficiency. Vector production anddetermination of GFP positive cells were as in FIG. 2. Mutation C wasfound not to affect the transduction efficacy of the vector whereasMutation A decreases the apparent titer of the vector by a factor 10.However, the lower number of GFP positive cells with the Mut A vectorreflects the fact that the mutation prevents polyadenylation at theviral LTR but does not indicate a low transduction efficacy. This pointis demonstrated in FIG. 7.

FIG. 6. Since the presence of MutA inhibits the function of the viralpolyadenylation signal, the MutA was tested in a vector carrying its ownpolyadenylation signal, pA, and the GFP gene. Mut A vectors carryingtheir own polyadenylation signal function as wild-type vectors.

FIG. 7. Apparent titers of wild-type and vectors carrying MutA and apolyadenylation signal (pA) were identical. Vector production anddetermination of GFP positive cells were as in FIG. 2.

FIG. 8. Sequence and secondary structure of mutations A and C with theentire panel of six disclosed by Das et al. (1997).

FIG. 9. Infectivity of wild-type HIV-1 in HeLa cells. Colored cellsindicate successful infection. Each colored cell corresponds to oneinfection event.

FIG. 10. Substantially reduced infectivity conferred by an inactivecPPT/cTS region in conjunction with a wild-type genome length. Viraltiters were adjusted so as to equalize reverse transcriptase activity tothose used in FIG. 9.

FIG. 11. Infectivity conferred by an inactive cPPT/cTS region inconjunction with a genome length 1470 shorter than wild type. Viraltiters were adjusted so as to equalize reverse transcriptase activity tothose used in FIGS. 9 and 10.

FIG. 12. Background staining of cells in the absence of virus. Theabsence of colored cells indicates the lack of false positives in theassays that produced FIGS. 9, 10, and 11.

SEQUENCE SUMMARY

SEQ ID NO:1 corresponds to positions 5296 to 5760 of the plasmid pMDLg/p RRE, derived from the HIV-1 molecular clone NL4-3 (Accession numberM19921) but modified to inactivate the cPPT/cTS region. The resultingsequence differs from the wild-type in the cPPT/cTS region, positions5432 through 5452 as indicated in FIG. 1 and described in SEQ ID NO:4.SEQ ID NO:2 and SEQ ID NO:3 correspond to nucleotide positions 5954through 6558, inclusive, of previously a described vector, pHR'-CMVLacZ,(Accession number AF105229), but incorporate the nucleotide sequencechanges as described by Das, et al. (1997). The sequences contain Eco RVand Bss HII restriction enzyme sites at the 5′ and 3′ ends,respectively, which are useful in introducing the sequences into desiredconstructs. SEQ ID NO:5 and SEQ ID NO:6 are the sequences of the poly(A)hairpin structures that substantially inhibit viral replication asidentified in FIG. 8 and described in Das, et al. (1997), and which arecontained within SEQ ID NO:2 and SEQ ID NO:3, respectively.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

While lentiviral vectors offer a great potential for gene-therapy andespecially the transduction of human hematopoietic stem cells (hHSC),vectors developed so far still suffer from concerns regarding theirbiosafety. The present invention overcomes such and other deficienciesin the art and describes the development of HIV-derived vectors thathave improved biosafety characteristics.

The present invention provides HIV-derived vectors which are safe,highly efficient, and very potent for expressing transgenes in human andanimal cells, including but not limited to hematopoietic progenitorcells as well as in all other blood cell derivatives. These vectorstherefore provide useful tools for genetic treatments such as inheritedand acquired disorders, gene-therapies for cancers especially thehematological cancers, as well as for the study of hematopoiesis vialentivector-mediated modification of human HSCs.

A. LENTIVIRAL VECTORS AND GENE THERAPY

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. The higher complexity enables the virus tomodulate its life cycle, as in the course of latent infection. Someexamples of lentivirus include the Human Immunodeficiency Viruses:HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviralvectors have been generated by multiply attenuating the HIV virulencegenes, for example, the genes env, vif, vpr, vpu, nef and tat aredeleted making the vector biologically more safe.

Lentiviral vectors offer great advantages for gene therapy. Theyintegrate stably into chromosomes of target cells which is required forlong-term expression. Further, they do not transfer viral genestherefore avoiding the problem of generating transduced cells that canbe destroyed by cytotoxic T-cells. Furthermore, they have a relativelylarge cloning capacity, sufficient for most envisioned clinicalapplications. In addition, lentiviruses, in contrast to otherretroviruses, are capable of transducing non-dividing cells. This isvery important in the context of gene-therapy for tissues such as thehematopoietic system, the brain, liver, lungs and muscle. For example,vectors derived from HIV-1 allow efficient in vivo and ex vivo delivery,integration and stable expression of transgenes into cells such aneurons, hepatocytes, and myocytes (Blomer et al., 1997; Kafri et al.,1997; Naldini et al., 1996; Naldini et al., 1998).

The lentiviral genome and the proviral DNA have the three genes found inretroviruses: gag, pol and env, which are flanked by two long terminalrepeat (LTR) sequences. The gag gene encodes the internal structural(matrix, capsid and nucleocapsid) proteins; the pol gene encodes theRNA-directed DNA polymerase (reverse transcriptase), a protease and anintegrase; and the env gene encodes viral envelope glycoproteins. The 5′and 3′ LTR's serve to promote transcription and polyadenylation of thevirion RNAs, respectively. Lentiviruses have additional genes includingvif, vpr, tat, rev, vpu, nef and vpx.

Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsidation of viral. RNA into particles (the Psi site). If thesequences necessary for encapsidation (or packaging of retroviral RNAinto infectious virions) are missing from the viral genome, the cisdefect prevents encapsidation of genomic RNA. However, the resultingmutant remains capable of directing the synthesis of all virionproteins.

Lentiviral vectors are known in the art, see Naldini et al., (1996a,1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat.Nos. 6,013,516; and 5,994,136 all incorporated herein by reference. Ingeneral, these vectors are plasmid-based or virus-based, and areconfigured to carry the essential sequences for incorporating foreignnucleic acid, for selection and for transfer of the nucleic acid into ahost cell.

Two components are involved in making a virus-based gene deliverysystem: first, the packaging elements, encompassing the structuralproteins as well as the enzymes necessary to generate an infectiousparticle, and second, the vector itself, i.e., the genetic material tobe transferred. Biosaftey safeguards can be introduced in the design ofboth of these components. Thus, the packaging unit of the firstgeneration HIV-based vectors comprised all HIV-1 proteins except theenvelope protein (Naldini et al., 1998, 1996a). Subsequently it wasshown that the deletion of four additional viral genes that areresponsible for virulence including, vpr, vif, vpu and nef did not alterthe utility of the vector system (Zufferey et al., 1997). It was alsoshown that Tat, the main transactivator of HIV is also dispensable forthe generation of a fully efficient vector (Dull et al., 1998). Thus,the third-generation packaging unit of the HIV-based lentiviral vectorscomprise only three genes of the parental virus: gag, pol and rev, whichhelps to eliminate the possibility of reconstitution of a wild-typevirus through recombination.

This system was further improved by removing HIV transcriptional unitsfrom the vector (Zufferey et al., 1998). It was demonstrated thereinthat introducing a deletion in the U3 region of the 3′ LTR of the DNAused to produce the vector RNA generated self-inactivating (SIN)vectors. During reverse transcription this deletion is transferred tothe 5′ LTR of the proviral DNA. Enough sequence was eliminated,including the removal of a TATA box, which abolished the transcriptionalactivity of the LTR, which prevents production of full-length vector RNAin transduced cells. This however did not affect vector titers or the invitro or in vivo properties of the vector.

The present invention provides several improvements to the existinglentivectors as described above and in other parts of thisspecification. Introducing a lentivector providing a heterologous gene,such as genes to treat hematopoietic and lympho-hematopoietic disordersin this invention, into a packaging cell yields a producer cell whichreleases infectious vector particles carrying the foreign gene ofinterest.

The env gene can be derived from any virus, including retroviruses. Theenv preferably is an amphotropic envelope protein which allowstransduction of cells of human and other species. Examples ofretroviral-derived env genes include, but are not limited to: Moloneymurine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma virus(HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon apeleukemia virus (GaLV or GALV), human immunodeficiency virus (HIV) andRous sarcoma virus (RSV). Other env genes such as Vesicular stomatitisvirus (VSV) protein G (VSV G), that of hepatitis viruses and ofinfluenza also can be used.

While VSV G protein is a desirable env gene because VSV G confers broadhost range on the recombinant virus, VSV G can be deleterious to thehost cell, e.g. the packaging cell. Thus, when a gene such as that forVSV G is used, it is preferred to employ an inducible promoter system sothat VSV G expression can be regulated to minimize host toxicity whenVSV G is expression is not required. For example, thetetracycline-regulated gene expression system of Gossen & Bujard, (1992)can be employed to provide for inducible expression of VSV G whentetracycline is withdrawn from the transferred cell. Thus, the tet/VP16transactivator is present on a first vector and the VSV G codingsequence is cloned downstream from a promoter controlled by tet operatorsequences on another vector.

The vector providing the viral env nucleic acid sequence is associatedoperably with regulatory sequences, e.g., a promoter or enhancer. Theregulatory sequence can be any eukaryotic promoter or enhancer,including for example, EF1α, PGK, the Moloney murine leukemia viruspromoter-enhancer element, the human cytomegalovirus enhancer, thevaccinia P7.5 promoter or the like (also see examples listed in Tables 1and 2 below). In some cases, such as the Moloney murine leukemia viruspromoter-enhancer element, the promoter-enhancer elements are locatedwithin or adjacent to the LTR sequences. Preferably, the regulatorysequence is one which is not endogenous to the lentivirus from which thevector is being constructed. Thus, if the vector is being made from SIV,the SIV regulatory sequence found in the SIV LTR would be replaced by aregulatory element which does not originate from SIV.

One may further target the recombinant virus by linkage of the envelopeprotein with an antibody or a particular ligand for targeting to areceptor of a particular cell-type. By inserting a sequence (including aregulatory region) of interest into the viral vector, along with anothergene which encodes the ligand for a receptor on a specific target cell,for example, the vector is now target-specific. Retroviral vectors canbe made target-specific by inserting, for example, a glycolipid or aprotein. Targeting often is accomplished by using an antigen-bindingportion of an antibody or a recombinant antibody-type molecule, such asa single chain antibody, to target the retroviral vector. Those of skillin the art will know of, or can readily ascertain without undueexperimentation, specific methods to achieve delivery of a retroviralvector to a specific target.

The heterologous or foreign nucleic acid sequence, such as apolynucleotide sequence encoding a gene such as a therapeutic gene forinherited or acquired hematopoietic disorders herein, is linked operablyto a regulatory nucleic acid sequence. Preferably, the heterologoussequence is linked to a promoter, resulting in a chimeric gene.

Marker genes may be utilized to assay for the presence of the vector,and thus, to confirm infection and integration. The presence of a markergene ensures the selection and growth of only those host cells whichexpress the inserts. Typical selection genes encode proteins that conferresistance to antibiotics and other toxic substances, e.g., histidinol,puromycin, hygromycin, neomycin, methotrexate, and cell surface markers.

The recombinant virus of the invention is capable of transferring anucleic acid sequence into a mammalian cell. The term, “nucleic acidsequence”, refers to any nucleic acid molecule, preferably DNA, asdiscussed in detail herein. The nucleic acid molecule may be derivedfrom a variety of sources, including DNA, cDNA, synthetic DNA, RNA orcombinations thereof. Such nucleic acid sequences may comprise genomicDNA which may or may not include naturally occurring introns. Moreover,such genomic DNA may be obtained in association with promoter regions,poly A sequences or other associated sequences. Genomic DNA may beextracted and purified from suitable cells by means well known in theart. Alternatively, messenger RNA (mRNA) can be isolated from cells andused to produce cDNA by reverse transcription or other means.

The vectors are introduced via transfection or infection into thepackaging cell line. The packaging cell line produces viral particlesthat contain the vector genome. Methods for transfection or infectionare well known by those of skill in the art. After cotransfection of thepackaging vectors and the transfer vector to the packaging cell line,the recombinant virus is recovered from the culture media and titteredby standard methods used by those of skill in the art. Thus, thepackaging constructs can be introduced into human cell lines by calciumphosphate transfection, lipofection or electroporation, generallytogether with a dominant selectable marker, such as neomycin, DHFR,Glutamine synthetase or ADA, followed by selection in the presence ofthe appropriate drug and isolation of clones. The selectable marker genecan be linked physically to the packaging genes in the construct.

Stable cell lines wherein the packaging functions are configured to beexpressed by a suitable packaging cell are known. For example, see U.S.Pat. No. 5,686,279; and Ory et al., (1996), which describe packagingcells. The packaging cells with a lentiviral vector incorporated in themform producer cells. Producer cells are thus cells or cell-lines thatcan produce or release packaged infectious viral particles carrying thetherapeutic gene of interest. These cells can further be anchoragedependent which means that these cells will grow, survive, or maintainfunction optimally when attached to a surface such as glass or plastic.The producer cells may also be neoplastically transformed cells. Someexamples of anchorage dependent cell lines used as lentiviral vectorpackaging cell lines when the vector is replication competent are HeLaor 293 cells and PERC.6 cells.

In some applications, particularly when the virus is to be used for genetherapy applications, it is preferable that the vector be replicationdeficient (or replication defective) to avoid uncontrolled proliferationof the virus in the individual to be treated. In such instancesmammalian cell lines are selected which have been engineered, either bymodification of the producer cell's genome to encode essential viralfunctions or by the co-infection of the producer cell with a helpervirus, to express proteins complementing the effect of the sequencesdeleted from the viral genome. For example, for HIV-1 derived vectors,the HIV-1 packaging cell line, PSI422, may be used as described inCorbeau, et al. (1996). Similarly, where the viral vector to be producedis a retrovirus, the human 293-derived retroviral packaging cell line(293GPG) capable of producing high titers of retroviral particles may beemployed as described in Ory, et al. (1996). In the production ofminimal vector systems, the producer cell is engineered (either bymodification of the viral genome or by the use of helper virus orcosmid) to complement the functions of the parent virus enablingreplication and packaging into virions in the producer cell line.

Lentiviral transfer vectors Naldini et al., (1996), have been used toinfect human cells growth-arrested in vitro and to transduce neuronsafter direct injection into the brain of adult rats. The vector wasefficient at transferring marker genes in vivo into the neurons and longterm expression in the absence of detectable pathology was achieved.Animals analyzed ten months after a single injection of the vectorshowed no decrease in the average level of transgene expression and nosign of tissue pathology or immune reaction (Blomer et al., 1997).

B. THE cPPT/cTS REGION

The introduction of foreign nucleic acids into the nucleus of a cellrequires importation of the nucleic acids into the nucleus through thenuclear membrane. Lentiviruses utilize an active nuclear import system,which forms the basis of their ability to replicate efficiently innon-dividing cells. This active import system relies upon a complexseries of events including a specific modality for reversetranscription. In particular, in HIV-1, the central polypurine tract(cPPT), located within the pol gene, initiates synthesis of a downstreamplus strand while plus strand synthesis is also initiated at the 3′polypurine tract (PPT). After strand transfer of the short DNA molecule,the upstream plus strand synthesis will initiate and proceed until thecenter of the genome is reached. At the central termination sequence(cTS) the HIV-1 reverse transcriptase is ejected, (released from itstemplate), when functioning in a strand displacement mode. (Charneau, etal., 1994) The net result is a double stranded DNA molecule with astable flap, 99 nucleotides in length at the center of the genome.

This central “flap” facilitates nuclear import. (Zennou, et al., 2000).Defects in the cPPT/cTS region that prevent the efficient reversetranscription initiating at the cPPT/cTS region prevent the formation ofthe central DNA flap. The resulting DNA molecules accumulate asnon-integrated linear viral DNA outside the nucleus. (Zennou, et al.,2000). Thus, an inactive, or substantially less active cPPT/cTS regionin a lentiviral vector packaging construct, if reconstituted into anRCR, will prevent efficient nuclear import of the RCR DNA genome duringany subsequent steps towards infection. The absence of a DNA flap in anHIV-1 virus system severely impairs viral DNA nuclear import. (Zennou,et al., 2000). Importantly, Zennou, et al. show that the addition ofcPPT on the transfer vector increases levels of integration by a factorof five, whereas the inactivation of the cPPT on the viral genome itselfdecreases replication by several order of magnitude. Although unknown toZennou, et al., this difference is a function of the shorter size of thevector compared to the viral genome.

The cPPT/cTS region acts in cis with the rest of the viral genome. Theregion extends over 118 nucleotides in HIV-1 and exists in similar formin other lentiviruses. The region is found at or near the center of alllentiviral genomes (Zennou, et al., 2000). The cPPT/cTS sequence elementoverlaps with the gene encoding the integrase protein and is present inan active form in all packaging systems described to date.

Wild-type activity of the cPPT/cTS region may be effectively eliminatedby the mutation of the underlying nucleic acid sequence so as to disruptthe activity without effecting the function of the integrase protein,which is also encoded by that sequence and its surrounding sequence.Packaging plasmids so altered do not reduce the vector titers that maybe achieved and so retain all the benefits of any vector productionsystem in which they are incorporated.

The elimination of wild-type activity of the cPPT/cTS region from viralpackaging systems improves their biosafety by preventing the efficientnuclear import of any RCR DNA genome during any subsequent steps towardsinfection. This protective effect of an inactive cPPT/cTS region mayoperate in any RCR lentiviral genome. However, the protective effectscan be optimized or enhanced by incorporating into the packaging plasmida stuffer sequence, whose purpose is to enlarge the eventual genome sizeof any RCR that may be produced. Larger viral genomes are more dependentupon a fully functional cPPT/cTS region for entry into the nucleus.Thus, a larger genome size, at least the size of a wild-type lentivirussuch as HIV-1, is less able to enter the nucleus through the mechanismmediated by the cPPT/cTS sequence region. Correspondingly, in lentiviralvectors packaging plasmids whose size has been shortened through theremoval or modification of non-essential or virulence encoding genes, astuffer sequence may be inserted to enlarge the genome size, thusutilizing more effectively the protective effects of an inactive ormutant cPPT/cTS region.

The stuffer sequence need not be of any particular sequence other thanone which does not rescue infectivity or in any other way contribute tovirulence of any possible RCRs that might be generated. The sequenceshould be of a size, however, to increase the protective effects ofinactive or mutant cPPT/cTS regions. For a minimal packaging plasmidsuch as pMDLD a stuffer sequence of about 4.4 kb in size effectivelyrecreates the native genome length of a lentivirus, and thus effectivelyaugments the effects of mutant cPPT/cTS regions. Optimally, the stuffersequence will be located between the pol gene and the RRE, a locationthat optimizes the likely effects of a larger genome size on theinhibition of nuclear import by mutant cPPT/cTS regions.

C. DRUG SUSCEPTIBILITY

The biosafety benefits provided by the replication inhibitory effects oflarger RCR genomes in conjunction with inactive cPPT/cTS regions may befurther enhanced by employing drug susceptibility genes. Drugsusceptibility genes encode proteins whose presence results in any virusincorporating/expressing the genes being susceptible to therapeuticdrugs. Thus, any unintended RCR infection may be specifically andeffectively treated.

The stuffer sequence may encode such drug susceptibility genes. Oneparticular sequence that confers drug susceptibility is the thymidinekinase gene (Zhao-Emonet et al., 1999). The expression of a drugsusceptibility gene such as thymidine kinase may be driven by apromoter. One such promoter is the IRES element. Further details of theIRES element and its use as a promoter is provided below. In the currentcontext, the IRES promoter and a thymidine kinase gene may be providedas an expression cassette, which may be inserted into the packagingplasmid as the “IRES-tk” cassette. The insertion of the IRES-tk cassetteprovides both for a genome length that aids in the effectiveness of themodifications to the cPPT/cTS region and provides a “suicide” gene thatallows therapeutic treatment of any infection with RCRs (one treat theinfected patient, not the RCR itself) that are produced and infective(Zhao-Emonet et al., 1999). The tk gene of the IRES-tk cassette isderived from the Herpes simplex virus and confers susceptibility to thedrug Ganciclovir, a substrate of TK. Thus, if an RCR is generated thatis capable of infection, the resulting infected cells may be killed withGanciclovir, thereby preventing the further spread of the RCR.Similarly, cells expressing the cytosine deaminase gene can be killedwith 5-fluorocytosine (Greco, 2001).

D. THE POLY(A) HAIRPIN

The 5′ untranslated leader sequences of lentiviral genomes containseveral sequence elements crucial for viral replication. These includeelements essential for transcription, mRNA splicing, dimerization,packaging, and reverse transcription. Much of the function of theregions depends upon the secondary structure of the viral RNA (Das, etal., 1997). One such structure is a hairpin that comprises thepolyadenylation signal (AAUAAA). The structure is therefore known as thepoly(A) hairpin. The poly(A) hairpin is part of the R-U5 domain of theLTR and is present in both the 5′ and 3′ ends of the proviral genome oflentiviruses.

The role of the poly(A) hairpin in replication activity has beenconserved despite the divergence in sequence among the variouslentiviruses (Das, et al., 1997). Disruption of the poly(A) hairpinstructure through mutation of the sequence involved severely inhibitsreplication activity (Das, et al., 1997). Mutant sequences of the 5′ LTRpoly(A) hairpin region can induce such defects in replication if theyare such that they either sufficiently destabilize the hairpin or act toexcessively stabilize the hairpin. For an efficient hairpin structure,the thermodynamic stability of the sequence pairings must remain withina relatively narrow limits (Das, et al., 1997).

Das, et al., (1997), incorporated herein by reference, created severaldifferent mutations within the 5′ LTR poly(A) region of HIV-1 andevaluated the effects of those sequence mutations on replicationactivity. Mutant A of Das, et al. (1997) stabilized the hairpinstructure to an extent sufficient to substantially inhibit wild-typereplication activity. Mutant C of Das, et al. (1997) destabilized thehairpin structure and also substantially inhibited replication activity.The sequences of Mutant A and Mutant C are provided herein as SEQ IDNO:2 and SEQ ID NO:5 for Mutant A, and SEQ ID NO:3 and SEQ ID NO:6 forMutant C, respectively.

Particular embodiments of the present invention may include providing atransfer vector incorporating the replication inhibiting 5′ LTR poly(A)sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:6.Preferably, these sequence elements are present in combination with oneor more aspects of the embodiments described elsewhere in thisspecification. Thus, mutant 5′ LTR poly(A) sequences may be incorporatedinto a transfer vector that is to be used in conjunction with apackaging plasmid incorporating the cPPT/cTS region displaying reducedreplication activity. Further, the transfer vector of the presentinvention may be used in conjunction with a packaging plasmid containinga stuffer sequence to further maximize the effects of the mutatedcPPT/cTS regions so employed. As indicated elsewhere in thisspecification, such a stuffer sequence may encode drug susceptibilitygenes or expression cassettes for drug susceptibility. All these aspectsof the present invention will be present in a most preferred embodimentof the present invention.

E. SIN DESIGN

The SIN design further increases the biosafety of lentiviral vectors. Amajority of the HIV LTR is comprised of the U3 sequences. The U3 regioncontains the enhancer and promoter elements that modulate basal andinduced expression of the HIV genome in infected cells and in responseto cell activation. Several of these promoter elements are essential forviral replication. Some of the enhancer elements are highly conservedamong viral isolates and have been implicated as critical virulencefactors in viral pathogenesis. The enhancer elements may act toinfluence replication rates in the different cellular target of thevirus (Marthas et al., 1993).

As viral transcription starts at the 3′ end of the U3 region of the 5′LTR, those sequences are not part of the viral mRNA and a copy thereoffrom the 3′ LTR acts as template for the generation of both LTR's in theintegrated provirus. If the 3′ copy of the U3 region is altered in aretroviral vector construct, the vector RNA is still produced from theintact 5′ LTR in producer cells, but cannot be regenerated in targetcells. Transduction of such a vector results in the inactivation of bothLTR's in the progeny virus. Thus, the retrovirus is self-inactivating(SIN) and those vectors are known as SIN transfer vectors.

The SIN design is described in further detail in Zufferey et al., 1998and U.S. Pat. No. 5,994,136 both incorporated herein by reference. Asdescribed therein, there are, however, limits to the extent of thedeletion at the 3′ LTR. First, the 5′ end of the U3 region servesanother essential function in vector transfer, being required forintegration. Thus, the terminal dinucleotide and att sequence mayrepresent the 5′ boundary of the U3 sequences which can be deleted. Inaddition, some loosely defined regions may influence the activity of thedownstream polyadenylation site in the R region. Excessive deletion ofU3 sequence from the 3′ LTR may decrease polyadenylation of vectortranscripts with adverse consequences both on the titer of the vector inproducer cells and the transgene expression in target cells. On theother hand, limited deletions may not abrogate the transcriptionalactivity of the LTR in transduced cells.

The lentiviral vectors described herein carry deletions of the U3 regionof the 3′ LTR spanning from nucleotide −418 to −18. This is the mostextensive deletion and extends as far as to the TATA box, thereforeabrogating any transcriptional activity of the LTR in transduced cells.The titer of vector in producer cells as well as transgene expression intarget cells was unaffected in these vectors. This design thereforeprovides an enormous increase in vector safety.

SIN-type vectors with such extensive deletions of the U3 region cannotbe generated for murine leukemia virus (MLV) or spleen necrosis virus(SNV) based retroviral vectors without compromising efficiency oftransduction.

Elimination of the −418 to −18 nucleotide sequence abolishestranscriptional activity of the LTR, thereby abolishing the productionof full length vector RNA in transduced cells. In the HIV-derivedlentivectors none of the in vitro or in vivo properties were compromisedby the SIN design. Importantly, the additional biosafety features of thepresent invention may be incorporated into SIN-type vectors andnon-SIN-type vectors with equal results.

G. POSTTRANSCRIPTIONALLY REGULATING ELEMENTS (PRE)

Enhancing transgene expression may be required in certain embodiments,especially those that involve lentiviral constructs of the presentinvention with modestly active promoters.

One type of PRE is an intron positioned within the expression cassette,which can stimulate gene expression. However, introns can be spliced outduring the life cycle events of a lentivirus. Hence, if introns are usedas PRE's they may have to be placed in an opposite orientation to thevector genomic transcript.

Posttranscriptional regulatory elements that do not rely on splicingevents offer the advantage of not being removed during the viral lifecycle. Some examples are the posttranscriptional processing element ofherpes simplex virus, the posttranscriptional regulatory element of thehepatitis B virus (HPRE) and the woodchuck hepatitis virus (WPRE). Ofthese the WPRE is most preferred as it contains an additional cis-actingelement not found in the HPRE (Donello et al., 1998). This regulatoryelement is positioned within the vector so as to be included in the RNAtranscript of the transgene, but downstream of stop codon of thetransgene translational unit. As demonstrated in the present inventionand in Zufferey et al., 1999, the WPRE element is a useful tool forstimulating and enhancing gene expression of desired transgenes in thecontext of the lentiviral vectors.

The WPRE is characterized and described in U.S. Pat. No. 6,136,597,incorporated herein by reference. As described therein, the WPRE is anRNA export element that mediates efficient transport of RNA from thenucleus to the cytoplasm. It enhances the expression of transgenes byinsertion of a cis-acting nucleic acid sequence, such that the elementand the transgene are contained within a single transcript. Presence ofthe WPRE in the sense orientation was shown to increase transgeneexpression by up to 7 to 10 fold. Retroviral vectors deliver sequencesin the form of cDNAs instead of complete intron-containing genes asintrons are generally spliced out during the sequence of events leadingto the formation of the retroviral particle. Introns mediate theinteraction of primary transcripts with the splicing machinery. Becausethe processing of RNAs by the splicing machinery facilitates theircytoplasmic export, due to a coupling between the splicing and transportmachineries, cDNAs are often inefficiently expressed. Thus, theinclusion of the WPRE in a vector results in enhanced expression oftransgenes.

H. NUCLEIC ACIDS

One embodiment of the present invention is to transfer nucleic acidsencoding a therapeutic gene, especially a gene that provides therapy forhematopoietic and lympho-hematopoietic disorders, such as the inheritedor acquired disorders described above. In one embodiment the nucleicacids encode a full-length, substantially full-length, or functionalequivalent form of such a gene.

Thus, in some embodiments of the present invention, the treatment of ahematopoietic and lympho-hematopoietic disorder involves theadministration of a lentiviral vector of the invention comprising atherapeutic nucleic acid expression construct to a cell of hematopoieticorigin. It is contemplated that the hematopoietic cells take up theconstruct and express the therapeutic polypeptide encoded by nucleicacid, thereby restoring the cells normal phenotype.

A nucleic acid may be made by any technique known to one of ordinaryskill in the art. Non-limiting examples of synthetic nucleic acid,particularly a synthetic oligonucleotide, include a nucleic acid made byin vitro chemical synthesis using phosphotriester, phosphite orphosphoramidite chemistry and solid phase techniques such as describedin EP 266,032, incorporated herein by reference, or via deoxynucleosideH-phosphonate intermediates as described by Froehler et al., 1986, andU.S. Pat. No. 5,705,629, each incorporated herein by reference. Anon-limiting example of enzymatically produced nucleic acid include oneproduced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of oligonucleotidesdescribed in U.S. Pat. No. 5,645,897, incorporated herein by reference.A non-limiting example of a biologically produced nucleic acid includesrecombinant nucleic acid production in living cells (see for example,Sambrook et al. 1989, incorporated herein by reference).

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, or by any other means known to one of ordinaryskill in the art (see for example, Sambrook et al. 1989, incorporatedherein by reference).

The term “nucleic acid” will generally refer to at least one molecule orstrand of DNA, RNA or a derivative or mimic thereof, comprising at leastone nucleobase, such as, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., adenine “A,” guanine “G,” thymine“T,” and cytosine “C”) or RNA (e.g. A, G, uracil “U,” and C). The term“nucleic acid” encompasses the terms “oligonucleotide” and“polynucleotide.” The term “oligonucleotide” refers to at least onemolecule of between about 3 and about 100 nucleobases in length. Theterm “polynucleotide” refers to at least one molecule of greater thanabout 100 nucleobases in length. These definitions generally refer to atleast one single-stranded molecule, but in specific embodiments willalso encompass at least one additional strand that is partially,substantially or fully complementary to the at least one single-strandedmolecule. Thus, a nucleic acid may encompass at least onedouble-stranded molecule or at least one triple-stranded molecule thatcomprises one or more complementary strand(s) or “complement(s)” of aparticular sequence comprising a strand of the molecule.

In certain embodiments, a “gene” refers to a nucleic acid that istranscribed. As used herein, a “gene segment” is a nucleic acid segmentof a gene. In certain aspects, the gene includes regulatory sequencesinvolved in transcription, or message production or composition. Inparticular embodiments, the gene comprises transcribed sequences thatencode for a protein, polypeptide or peptide. In other particularaspects, the gene comprises a nucleic acid, and/or encodes a polypeptideor peptide-coding sequences of a gene that is defective or mutated in ahematopoietic and lympho-hematopoietic disorder. In keeping with theterminology described herein, an “isolated gene” may comprisetranscribed nucleic acid(s), regulatory sequences, coding sequences, orthe like, isolated substantially away from other such sequences, such asother naturally occurring genes, regulatory sequences, polypeptide orpeptide encoding sequences, etc. In this respect, the term “gene” isused for simplicity to refer to a nucleic acid comprising a nucleotidesequence that is transcribed, and the complement thereof. In particularaspects, the transcribed nucleotide sequence comprises at least onefunctional protein, polypeptide and/or peptide encoding unit. As will beunderstood by those in the art, this functional term “gene” includesboth genomic sequences, RNA or cDNA sequences, or smaller engineerednucleic acid segments, including nucleic acid segments of anon-transcribed part of a gene, including but not limited to thenon-transcribed promoter or enhancer regions of a gene. Smallerengineered gene nucleic acid segments may express, or may be adapted toexpress using nucleic acid manipulation technology, proteins,polypeptides, domains, peptides, fusion proteins, mutants and/or suchlike. Thus, a “truncated gene” refers to a nucleic acid sequence that ismissing a stretch of contiguous nucleic acid residues.

Various nucleic acid segments may be designed based on a particularnucleic acid sequence, and may be of any length. By assigning numericvalues to a sequence, for example, the first residue is 1, the secondresidue is 2, etc., an algorithm defining all nucleic acid segments canbe created:

n to n+y

where n is an integer from 1 to the last number of the sequence and y isthe length of the nucleic acid segment minus one, where n+y does notexceed the last number of the sequence. Thus, for a 10-mer, the nucleicacid segments correspond to bases 1 to 10, 2 to 11, 3 to 12 . . . and/orso on. For a 15-mer, the nucleic acid segments correspond to bases 1 to15, 2 to 16, 3 to 17 . . . and/or so on. For a 20-mer, the nucleicsegments correspond to bases 1 to 20, 2 to 21, 3 to 22 . . . and/or soon.

The nucleic acid(s) of the present invention, regardless of the lengthof the sequence itself, may be combined with other nucleic acidsequences, including but not limited to, promoters, enhancers,polyadenylation signals, restriction enzyme sites, multiple cloningsites, coding segments, and the like, to create one or more nucleic acidconstruct(s). The overall length may vary considerably between nucleicacid constructs. Thus, a nucleic acid segment of almost any length maybe employed, with the total length preferably being limited by the easeof preparation or use in the intended recombinant nucleic acid protocol.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. Vectors of the present invention arelentivirus based as described above and in other parts of thespecification. The nucleic acid molecules carried by the vectors of theinvention encode therapeutic genes and will be used for carrying outgene-therapies. One of skill in the art would be well equipped toconstruct such a therapeutic vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described below.

(a) Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30-110 by upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 by apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include theβ-lactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well. Control sequences comprising promoters, enhancers andother locus or transcription controlling/modulating elements are alsoreferred to as “transcriptional cassettes”.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al., 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousfor gene therapy or for applications such as the large-scale productionof recombinant proteins and/or peptides. The promoter may beheterologous or endogenous.

Use of a T3, T7 or SP6 cytoplasmic expression system is another possibleembodiment. Eukaryotic cells can support cytoplasmic transcription fromcertain bacterial promoters if the appropriate bacterial polymerase isprovided, either as part of the delivery complex or as an additionalgenetic expression construct.

Tables 1 lists non-limiting examples of elements/promoters that may beemployed, in the context of the present invention, to regulate theexpression of a RNA. Table 2 provides non-limiting examples of inducibleelements, which are regions of a nucleic acid sequence that can beactivated in response to a specific stimulus.

TABLE 1 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983;Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-Dra Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnsonet al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOmitz et al., 1987 Metallothionein (MTII) Karin et al., 1987; Culotta etal., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987 AlbuminPinkert et al., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godboutet al., 1988; Campere et al., 1989 γ-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM)α₁-Antitrypsin Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-RegulatedProteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al.,1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerjiet al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al.,1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wanget al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinkaet al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbelland/or Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983;Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze etal., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al.,1988; Celander et al., 1988; Chol et al., 1988; Reisman et al., 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/orWilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al.,1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al.,1987; Spandau et al., 1988; Vannice et al., 1988 Human ImmunodeficiencyVirus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al.,1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddocket al., 1989 Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn etal., 1989

TABLE 2 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Haslinger et Heavy metals al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse mammaryGlucocorticoids Huang et al., 1981; Lee et tumor virus) al., 1981;Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta etal., 1985; Sakai et al., 1988 β-Interferon Poly(rI)x Tavernier et al.,1983 Poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984 CollagenasePhorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA)Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b MurineMX Gene Interferon, Newcastle Hug et al., 1988 Disease Virus GRP78 GeneA23187 Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2κb InterferonBlanar et al., 1989 HSP70 ElA, SV40 Large T Taylor et al., 1989, 1990a,1990b Antigen Proliferin Phorbol Ester-TPA Mordacq et al., 1989 TumorNecrosis Factor PMA Hensel et al., 1989 Thyroid Stimulating ThyroidHormone Chatterjee et al., 1989 Hormone α Gene

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Non-limiting examples of such regions include the human LIMK2 gene(Nomoto et al., 1999), the somatostatin receptor 2 gene (Kraus et al.,1998), murine epididymal retinoic acid-binding gene (Lareyre et al.,1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen(Tsumaki, et al., 1998), DIA dopamine receptor gene (Lee, et al., 1997),insulin-like growth factor II (Wu et al., 1997), and human plateletendothelial cell adhesion molecule-1 (Almendro et al., 1996).

The lentiviral vectors of the present invention are designed, primarily,to transfect cells with a therapeutic gene under the control ofregulated eukaryotic promoters. Although the EF1 α-promoter and the PGKpromoter are preferred other promoter and regulatory signal elements asdescribed in the Tables 1 and 2 above may also be used. Additionally anypromoter/enhancer combination (as per the Eukaryotic Promoter Data BaseEPDB) could also be used to drive expression of structural genesencoding the therapeutic gene of interest that is used in context withthe lentiviral vectors of the present invention. Alternatively, atissue-specific promoter for cancer gene therapy or the targeting oftumors may be employed with the lentiviral vectors of the presentinvention for treatment of cancers, especially hematological cancers.

Typically promoters and enhancers that control the transcription ofprotein encoding genes in eukaryotic cells are composed of multiplegenetic elements. The cellular machinery is able to gather and integratethe regulatory information conveyed by each element, allowing differentgenes to evolve distinct, often complex patterns of transcriptionalregulation.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Aside from this operational distinction, enhancers and promoters arevery similar entities.

Promoters and enhancers have the same general function of activatingtranscription in the cell. They are often overlapping and contiguous,often seeming to have a very similar modular organization. Takentogether, these considerations suggest that enhancers and promoters arehomologous entities and that the transcriptional activator proteinsbound to these sequences may interact with the cellular transcriptionalmachinery in fundamentally the same way.

A signal that may prove useful is a polyadenylation signal (hGH, BGH,SV40). The use of internal ribosome binding sites (IRES) elements areused to create multigene, or polycistronic, messages. IRES elements areable to bypass the ribosome scanning model of 5′-methylatedcap-dependent translation and begin translation at internal sites(Pelletier and Sonenberg, 1988). IRES elements from two members of thepicornavirus family (polio and encephalomyocarditis) have been described(Pelletier and Sonenberg, 1988), as well as an IRES from a mammalianmessage (Macejak and Sarnow, 1991). IRES elements can be linked toheterologous open reading frames. Multiple open reading frames can betranscribed together, each separated by an IRES, creating polycistronicmessages. By virtue of the IRES element, each open reading frame isaccessible to ribosomes for efficient translation. Multiple genes can beefficiently expressed using a single promoter/enhancer to transcribe asingle message. In particular, the IRES element may be used to drive theexpression of drug susceptibility genes such as thymidine kinase and thelike.

In any event, it will be understood that promoters are DNA elementswhich when positioned functionally upstream of a gene leads to theexpression of that gene. Most transgenes that will be introduced usingthe lentiviral vectors of the present invention are functionallypositioned downstream of a promoter element.

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

(b) Multiple Cloning Sites

Vectors of the present invention can include a multiple cloning site(MCS), which is a nucleic acid region that contains multiple restrictionenzyme sites, any of which can be used in conjunction with standardrecombinant technology to digest the vector (see, for example,Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997,incorporated herein by reference.) “Restriction enzyme digestion” refersto catalytic cleavage of a nucleic acid molecule with an enzyme thatfunctions only at specific locations in a nucleic acid molecule. Many ofthese restriction enzymes are commercially available. Use of suchenzymes is widely understood by those of skill in the art. Frequently, avector is linearized or fragmented using a restriction enzyme that cutswithin the MCS to enable exogenous sequences to be ligated to thevector. “Ligation” refers to the process of forming phosphodiester bondsbetween two nucleic acid fragments, which may or may not be contiguouswith each other. Techniques involving restriction enzymes and ligationreactions are well known to those of skill in the art of recombinanttechnology.

(c) Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference.)

(d) Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to be more stable and are translated more efficiently. Thus,in other embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

(e) Polyadenylation Signals

In eukaryotic gene expression, one will typically include apolyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Some examples include the SV40 polyadenylationsignal or the bovine growth hormone polyadenylation signal, convenientand known to function well in various target cells. Polyadenylation mayincrease the stability of the transcript or may facilitate cytoplasmictransport.'

(f) Origins of Replication

In order to propagate a vector of the invention in a host cell, it maycontain one or more origins of replication sites (often termed “ori”),which is a specific nucleic acid sequence at which replication isinitiated. Alternatively an autonomously replicating sequence (ARS) canbe employed if the host cell is yeast.

(g) Selectable and Screenable Markers

In certain embodiments of the invention, cells transduced with thelentivectors of the present invention may be identified in vitro or invivo by including a marker in the expression vector. Such markers wouldconfer an identifiable change to the transduced cell permitting easyidentification of cells containing the expression vector. Generally, aselectable marker is one that confers a property that allows forselection. A positive selectable marker is one in which the presence ofthe marker allows for its selection, while a negative selectable markeris one in which its presence prevents its selection. An example of apositive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transfected cells, for example, genetic constructsthat confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT,zeocin and histidinol are useful selectable markers. In addition tomarkers conferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

I. HOST CELLS

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous nucleic acid encoded by the vectors of thisinvention. A host cell can, and has been, used as a recipient forvectors. A host cell may be “transfected” or “transformed,” which refersto a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A transformed cell includes the primarysubject cell and its progeny. As used herein, the terms “engineered” and“recombinant” cells or host cells are intended to refer to a cell intowhich an exogenous nucleic acid sequence, such as, for example, alentivector of the invention bearing a therapeutic gene construct, hasbeen introduced. Therefore, recombinant cells are distinguishable fromnaturally occurring cells which do not contain a recombinantlyintroduced nucleic acid.

In certain embodiments, it is contemplated that RNAs or proteinaceoussequences may be co-expressed with other selected RNAs or proteinaceoussequences in the same host cell. Co-expression may be achieved byco-transfecting the host cell with two or more distinct recombinantvectors. Alternatively, a single recombinant vector may be constructedto include multiple distinct coding regions for RNAs, which could thenbe expressed in host cells transfected with the single vector.

Host cells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (www.atcc.org). Some examples of host cells used inthis invention include but are not limited to virus packaging cells,virus producer cells, 293T cells, human hematopoietic progenitor cells,human hematopoietic stem cells, CD34⁺ cells, CD4⁺ cells, and the like.

(a) Tissues and Cells

A tissue may comprise a host cell or cells to be transformed orcontacted with a nucleic acid delivery composition and/or an additionalagent. The tissue may be part or separated from an organism. In certainembodiments, a tissue and its constituent cells may comprise, but is notlimited to, blood (e.g., hematopoietic cells, such as humanhematopoietic progenitor cells, human hematopoietic stem cells, CD34⁺cells, CD4⁺ cells, lymphocytes and other blood lineage cells), bonemarrow, brain, stem cells, blood vessel, liver, lung, bone, breast,cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial,epithelial, esophagus, facia, fibroblast, follicular, ganglion cells,glial cells, goblet cells, kidney, lymph node, muscle, neuron, ovaries,pancreas, peripheral blood, prostate, skin, small intestine, spleen,stomach, testes.

(b) Organisms

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, human,primate or murine. In other embodiments the organism may be anyeukaryote or even a prokayote (e.g., a eubacteria, an archaea), as wouldbe understood by one of ordinary skill in the art (see, for example,webpage http://phylogeny.arizona.edu/tree/phylogeny.html). Somelentivectors of the invention may employ control sequences that allowthem to be replicated and/or expressed in both prokaryotic andeukaryotic cells. One of skill in the art would further understand theconditions under which to incubate all of the above described host cellsto maintain them and to permit replication of a vector. Also understoodand known are techniques and conditions that would allow large-scaleproduction of the lentivectors of the invention, as well as productionof the nucleic acids encoded by the lentivectors and their cognatepolypeptides, proteins, or peptides some of which are therapeutic genesor proteins which will be used for gene therapies.

J. INJECTABLE COMPOSITIONS AND PHARMACEUTICAL FORMULATIONS

To achieve gene-therapy using the lentiviral vector compositions of thepresent invention, one would generally contact a cell in need thereofwith a lentiviral vector comprising a therapeutic gene. The cell willfurther be in an organism such as a human in need of the gene therapy.The routes of administration will vary, naturally, with the location andnature of the disease, and include, e.g., intravenous, intrarterial,intradermal, transdermal, intramuscular, intranasal, subcutaneous,percutaneous, intratracheal, intraperitoneal, intratumoral, perfusionand lavage. The cells will also sometimes be isolated from theorganisms, exposed to the lentivector ex vivo, and reimplantedafterwards.

Injection of lentiviral nucleic acid constructs of the invention may bedelivered by syringe or any other method used for injection of asolution, as long as the expression construct can pass through theparticular gauge of needle required for injection. A novel needlelessinjection system has recently been described (U.S. Pat. No. 5,846,233)having a nozzle defining an ampule chamber for holding the solution andan energy device for pushing the solution out of the nozzle to the siteof delivery. A syringe system has also been described for use in genetherapy that permits multiple injections of predetermined quantities ofa solution precisely at any depth (U.S. Pat. No. 5,846,225).

Solutions of the nucleic acids as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intraarterial, intramuscular, subcutaneous, intratumoral andintraperitoneal administration. In this connection, sterile aqueousmedia that can be employed will be known to those of skill in the art inlight of the present disclosure. For example, one dosage may bedissolved in 1 ml of isotonic NaCl solution and either added to 1000 mlof hypodermoclysis fluid or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition saltsand which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” or“pharmacologically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic lentiviral vectoris delivered to a target cell.

For gene therapy to discrete, solid, accessible tumors, intratumoralinjection, or injection into the tumor vasculature is specificallycontemplated. Local, regional or systemic administration also may beappropriate. For tumors of >4 cm, the volume to be administered will beabout 4-10 ml (preferably 10 ml), while for tumors of <4 cm, a volume ofabout 1-3 ml will be used (preferably 3 ml). Multiple injectionsdelivered as single dose comprise about 0.1 to about 0.5 ml volumes. Theviral particles may advantageously be contacted by administeringmultiple injections to the tumor, spaced at approximately 1 cmintervals. Systemic administration is preferred for conditions such ashematological malignancies.

Continuous administration also may be applied where appropriate.Delivery via syringe or catherization is preferred. Such continuousperfusion may take place for a period from about 1-2 hours, to about 2-6hours, to about 6-12 hours, to about 12-24 hours, to about 1-2 days, toabout 1-2 wk or longer following the initiation of treatment. Generally,the dose of the therapeutic composition via continuous perfusion will beequivalent to that given by a single or multiple injections, adjustedover a period of time during which the perfusion occurs.

Treatment regimens may vary as well, and often depend on type of diseaseand location of diseased tissue, and factors such as the health and theage of the patient. The clinician will be best suited to make suchdecisions based on the known efficacy and toxicity (if any) of thetherapeutic formulations based on lentiviral vectors of the presentinvention.

The treatments may include various “unit doses.” A unit dose is definedas containing a predetermined-quantity of the therapeutic compositioncomprising a lentiviral vector of the present invention. The quantity tobe administered, and the particular route and formulation, are withinthe skill of those in the clinical arts. A unit dose need not beadministered as a single injection but may comprise continuous infusionover a set period of time. Unit dose of the present invention mayconveniently be described in terms of transducing units (T.U.) oflentivector, as defined by titering the vector on a cell line such asHeLa or 293. Unit doses range from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, 10¹², 10¹³ T.U. and higher.

K. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Materials and Methodology Employed in Examples 1 through 3

Cell Lines and Culture Conditions

293T, F208 and Hela P4 cells were cultured in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calfserum, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/mlstreptomycin. Cell were cultured in incubators at 37° C. in a humidified5% CO₂ atmosphere.

Plasmids Construction

All plasmid modifications were done according to standard procedures(Sambrook et al. 1989).

Plasmid pHIV(BRU) contains the full-length proviral genome of HIV-1strain BRU. Plasmid pcPPT-D contains the full-length genome of HIV-1 butthe cPPT/cTS sequence element is mutated as described in SEQ ID NO:1 andSEQ ID NO:4.

Plasmid pHIV(BRU)ΔE was constructed by replacing the SalI-BamHI fragmentwith the corresponding fragment from pR9ΔE. Plasmid pcPPT-D ΔE wasconstructed similarly.

Plasmid pHIV(BRU)Δ 1470 was constructed by replacing the SalI-BamHIfragment with the corresponding fragment from pCMVΔR8.91. PlasmidpcPPT-D Δ 1470 was constructed similarly.

Vector Preparation

Stocks of vector were prepared as previously described (Zufferey, et al.1997, Zufferey, et al. 2000, incorporated herein by reference). Three orfour plasmids were transiently cotransfected into 293T cells to generatesecond and third generation lentiviral vector, respectively. Vectorpreparation and cell transduction were done in a BL2 laboratory. Reversetranscriptase activity was measured in each vector stock using themethod described in Klages et al. (2000), incorporated herein byreference. Differences in reverse transcriptase activity, usually lessthan 15%, were corrected by dilution of the stocks with high activity.

Virus Stocks Preparation

Stocks of virus were prepared by transfecting the different proviralplasmids into 293T cells. For pseudotyping experiments, envelopedefective proviral plasmid were cotransfected with the pMD.G plasmidencoding the VSV G protein. Reverse transcriptase activity was measuredin each virus stock using the method described in Klages et al. (2000).Differences in reverse transcriptase activity, usually less than 15%,were corrected by dilution of the stocks with high activity.

Vector Titration

Vectors were titrated on 293T and F208 cells. Target cell (5×10⁴cells/well) were plated in each well of a 6-well tissue culture plateand incubated 24 hour in 1 ml DMEM. Vector stocks (100 μl) or 4 serial1:10 dilutions was added to 1 ml of fresh DMEM and incubated for 2 moredays. Polybrene was omitted.

Flow Cytometry Analysis

Cells were analyzed as described (Arrighi et al., 1999), on aFACScalibur (Becton-Dickinson) with slight modifications. FL-1 was usedfor GFP, FL-2 for autofluorescence. Cells were fixed with 2%paraformaldehyde for 30 minutes, and resuspended into PBS prior toanalysis. Data were analyzed using WINMDI™ software written by J.Trotter at Scripps Institute (La Jolla, Calif.) and CellQuest software(Becton-Dickinson).

Virus Titration on HeLa P4 Cells

HeLa P4 cells express human CD4 and contain a reporter transgene made ofHIV-1 LTR fused to the E. coli LacZ gene. Upon HIV-1 infection andgenome integration, the HIV-1 Tat protein is produced. This proteintrans-activates the HIV-1 LTR promoter activity resulting in highexpression level of β-galactosidase encoded by the Lac Z gene.β-galactosidase activity is detected by an histochemical staining. HIV-1infected cells acquire a blue color. The histochemical detection ofβ-galactosidase is described in Zufferey, et al. (2000).

HeLa P4 cells (5×10⁴ cells/well) were plated in each well of a 6-welltissue culture plate and cultured in 1 ml DMEM for 24 hour before beinginfected. For infection, 1 ml of cPPT deficient vector stock was usedwhereas 1 ml and dilutions corresponding to 10, 5 and 1 μl were used forthe wild-type HIV-1. The number of blue foci were counted using aninverted light microscope in wells containing less than 100 infectionevents.

Example 1 Modification of a Lentiviral Packaging Plasmid CentralPolypurine Tract (cPPT/cTS)

Modifications to the sequence of the cPPT/cTS region may be made so thatnuclear import is severely hindered without interfering with theactivity of the pol gene of which the cPPT/cTS region is a part (Zennou,et al., 2000). Incorporated into a packaging plasmid, such sequenceswill be represented in any RCRs that may arise during the production oruse of lentiviral vectors and so will effectively inhibit the nuclearimport of these undesired RCRs. The packaging plasmid pMDLD incorporatesmodifications to the cPPT/cTS region that effectively inhibit nuclearimport of lentiviral genomes (FIG. 1).

Plasmid pMDLD is derived from pMDLg/pRRE, which has been described fullyelsewhere (Dull, et al., 1998, incorporated herein by reference).Briefly, pMDLg/pRRE is a CMV-driven expression plasmid that containsonly the gag and pol coding sequences from HIV-1. Additionally, a 374-bpRRE-containing sequence from HIV-1 (HXB2) is present immediatelydownstream of the pol coding sequences. An inactive cPPT/cTS region wassubstituted for that of pMDLD by replacing the wild-type AflII-BspEIfragment (positions 5296 to 5760 of the plasmid) with the correspondingAflII-BspEI fragment of SEQ ID NO: 1. The resulting sequence differs inthe cPPT/cTS region, positions 5432 through 5452 as indicated in FIG. 1and described in SEQ ID NO:4.

The mutations which inactivate the cPPT/cTS region do not impact on thefunction of the integrase protein. To test whether the novel packagingsystems have conserved their ability to produce HIV-1 vectors, vectorproduction by systems with an active or an inactive cPPT/cTS regionswere compared. Vectors encoding the Green Fluorescent Protein (GFP) weregenerated by transient co-transfection of 293T cells with four plasmidsaccording to previously published protocols (Zufferey, et al. 2000). Thetransfer vector used in these experiments was the transfer vectorplasmid pRRLCMV GFP SIN. The envelope plasmid used was pMD.G, whichencodes the vesicular stomatitis virus G protein. The pRSVrev plasmidencoding the HIV-1 Rev protein was also used.

The resulting vector stocks were assayed for reverse transcriptaseactivity to eliminate any difference which could result from variabilityin transfection efficiency. Stocks with matched reverse transcriptaseactivity were titrated on 293T cells and F208 cells. For titration, 10⁵cells were plated in each well of 6-well plates and cultured in 1 ml ofmedium. 24 hours after plating, cells were transduced with 500microliters of vector stock or of serial dilutions of vector stocks.

The percentage of GFP-expressing cells was determined 48 hours later byFluorescence Activated Cell Sorting (FACS). With both cell lines, wefound that vector titers were independent from the functionality of thecPPT/cTS sequence element in the packaging plasmids (FIG. 2). Thus, thecPPT/cTS sequence element can be inactivated in plasmids for thepackaging of HIV-1 based vectors without any decrease in vector titersproduced. The increase in biosafety conferred by the modification can beeffectively incorporated into useful methods for the production oflentiviral vectors.

Example 2 Insertion of a Stuffer Sequence into the Packaging PlasmidEnhances Biosafety

The biosafety of modifications to the sequence of the cPPT/cTS region isenhanced when the overall length of any resultant RCR genome issufficiently large. Such an increase in size is obtained by inserting astuffer sequence into the packaging plasmid pMDLD described above.

To test whether the infectivity of the HIV-1 virus in the absence of anactive cPPT/cTS sequence element depends on viral genome size, we havegenerated HIV-1 proviral genomes of decreasing size by removing sequencestretches encoding the envelope protein or accessory proteins. Themissing genetic information was provided in trans to complement thedefective viruses. The relative infectivity of HIV 1 viruses withdifferent genome sizes was assayed on P4 cells.

The decreasing genome size did not affect the infectivity of the virusescontaining a cPPT/cTS sequence element. In contrast, the infectivity ofthe mutated viruses increased when the genome size was reduced. For eachgenome size, we performed pairwise comparisons of viruses with orwithout an active cPPT/cTS sequence element. For viruses of wild-typelength, we found that the HIV-1 virus with an inactive cPPT/cTS sequenceelement is 200 times less infectious than its wild-type counterpart. Forviruses shorter by 1470 nucleotides, the virus with the inactivecPPT/cTS sequence element is only 70 times less infectious than itswild-type counterpart.

The inhibitory effect on viral replication due to the absence of thecPPT/cTS function increases with the viral genome size. Consequently,the size of the packaging plasmids for the production of lentiviralvectors may be increased in order to maximize the safety improvementobtained by the inactivation of the cPPT/cTS sequence element. The sizeof the packaging plasmids can be increased by inserting DNA at differentpositions. The highest safety is obtained by inserting DNA between theend of gag/pol gene and the RRE sequence element because DNA inserted atthis position is most likely included in the genome of a putativerecombinant virus.

Example 3 Creation of Replication Inhibiting Mutant 5′ LTR poly(A)Hairpins

Some mutations in the 5′ R-U5 region of the Long Terminal Repeat (LTR)have profound inhibitory effects on virus replication (Das, et al.,1997). Two mutated 5′ LTR poly(A) hairpin sequences (mutA and mutC,corresponding to SEQ ID NO: 5 and SEQ ID NO: 6, respectively) wereselected from a panel of altered poly(A) hairpin sequences as disclosedby Das, et al., (1997), (see FIG. 8). These two mutants were chosenbecause they have the strongest inhibitory effect on virus replication.Previous partial characterization of these mutations suggested that themutations might affect a step of viral replication that is not requiredfor the vector function.

To test the effects of these mutant sequences, mutations A and C in theR region of the 5′ LTR were introduced into the plasmid pHR′CMV GFP SIN(Zufferey, et al., 1998) so as to replace the wild-type sequence. Vectorwas produced using the wild-type or mutated transfer vector plasmids incombination with pCMV ΔR8.91 as packaging plasmid and pMD.G as envelopeplasmid expressing the VSV G protein. The packaging plasmid pCMV ΔR8.91is an HIV-derived packaging construct, which encodes the HIV-1 Gag andPol precursors, as well as the regulatory proteins Tat and Rev (Zuffereyet al., 1997). Vectors stocks were produced by transient transfection of293T cells according to published protocol (Zufferey, et al., 1997),matched for reverse transcriptase activity and titrated on 293T and F208cells as described.

Substantially identical titers for wild-type and mutC vectors weredisplayed. For the mutA vector, titers were apparently reduced by afactor of 10. Since the mutation A abolishes the function of the viralpolyA addition signal, the comparison was repeated using wild-type andmutated versions of the pHR′pA-GFP-I-AC plasmid in which the GFPtransgene has it own polyA signal independent from the LTR. In thissetting the titers of all three vector stocks were identical. Thesemutations in the R region of LTR can severely impair the replication ofthe HIV-1 virus without affecting the production and the transductionefficiency of an HIV-1 derived vector.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references and those references provided above, to theextent that they provide exemplary procedural or other detailssupplementary to those set forth herein, are specifically incorporatedherein by reference.

-   U.S. Pat. No. 4,682,195-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 5,466,468-   U.S. Pat. No. 5,645,897-   U.S. Pat. No. 5,686,279-   U.S. Pat. No. 5,705,629-   U.S. Pat. No. 5,846,225-   U.S. Pat. No. 5,846,233-   U.S. Pat. No. 5,925,565-   U.S. Pat. No. 5,928,906-   U.S. Pat. No. 5,935,819-   U.S. Pat. No. 5,994,136-   U.S. Pat. No. 6,013,516-   U.S. Pat. No. 6,136,597-   EP 266,032-   Akkina, Walton, Chen, Li, Planelles, Chen, “High-efficiency gene    transfer into CD34+ cells with a human immunodeficiency virus type    1-based retroviral vector pseudotyped with vesicular stomatitis    virus envelope glycoprotein G,” J. Virol., 70:2581-2585, 1996.-   Almendro et al., “Cloning of the human platelet endothelial cell    adhesion molecule-1 promoter and its tissue-specific expression.    Structural and functional characterization,” J. Immunol.,    157(12):5411-5421, 1996.-   An, Wersto, Agricola, Metzger, Lu, Amado, Chen, Donahue, “Marking    and gene expression by a lentivirus vector in transplanted human and    nonhuman primate CD34(+) cells,”J. Virol., 74:1286-1295, 2000.-   Angel, Bauman, Stein, Dellus, Rahmsdorf, and Herrlich,    “12-O-tetradecanoyl-phorbol-13-acetate Induction of the Human    Collagenase Gene is Mediated by an Inducible Enhancer Element    Located in the 5′ Flanking Region,” Mol. Cell. Biol., 7:2256, 1987a.-   Angel, Imagawa, Chiu, Stein, Imbra, Rahmsdorf, Jonat, Herrlich, and    Karin, “Phorbol Ester-Inducible Genes Contain a Common cis Element    Recognized by a TPA-Modulated Trans-acting Factor,” Cell, 49:729,    1987b-   Arrighi, Hauser, Chapuis, Zubler, Kindler, “Long-term culture of    human CD34(+) progenitors with FLT3-ligand, thrombopoietin, and stem    cell factor induces extensive amplification of a CD34(−)CD14(−) and    CD34(−)CD14(+) dendritic cell precursor,” Blood, 93:2244-2252, 1999.-   Atchison and Perry, “Tandem Kappa Immunoglobulin Promoters are    Equally Active in the Presence of the Kappa Enhancer: Implications    for Model of Enhancer Function,” Cell, 46:253, 1986.-   Atchison and Perry, “The Role of the Kappa Enhancer and its Binding    Factor NF-kappa B in the Developmental Regulation of Kappa Gene    Transcription,” Cell, 48:121, 1987.-   Banerji et al., “Expression of a Beta-Globin Gene is Enhanced by    Remote SV40 DNA Sequences,” Cell, 27:299, 1981.-   Banerji, Olson, and Schaffner, “A lymphocyte-specific cellular    enhancer is located downstream of the joining region in    immunoglobulin heavy-chain genes,” Cell, 35:729, 1983.-   Berkhout, Silverman, and Jeang, “Tat Trans-activates the Human    Immunodeficiency Virus Through a Nascent RNA Target,” Cell, 59:273,    1989.-   Bhatia, Bonnet; Kapp, Wang, Murdoch, Dick, “Quantitative analysis    reveals expansion of human hematopoietic repopulating cells after    short-term ex vivo culture,” J. Exp. Med., 186:619-624, 1997.-   Blanar, Baldwin, Flavell, and Sharp, “A gamma-interferon-induced    factor that binds the interferon response sequence of the MHC class    I gene, H-2 Kb,” EMBO J., 8:1139, 1989.-   Blomer, Naldini, Kafri, Trono, Verma, Gage, “Highly efficient and    sustained gene transfer in adult neurons with a lentivirus    vector,” J. Virol., 71:6641-6649, 1997.-   Bodine and Ley, “An enhancer element lies 3′ to the human a gamma    globin gene,” EMBO J, 6:2997, 1987.-   Boshart, Weber, Jahn, Dorsch-Hasler, Fleckenstein, and Schaffner, “A    very strong enhancer is located upstream of an immediate early gene    of human cytomegalovirus,” Cell, 41:521, 1985.-   Bosze, Thiesen, and Charnay, “A transcriptional enhancer with    specificity for erythroid cells is located in the long terminal    repeat of the friend murine leukemia virus,” EMBO J., 5:1615, 1986.-   Braddock, Chambers, Wilson, Esnouf, Adams, Kingsman, and Kingsman,    “HIV-I Tat activates presynthesized RNA in the nucleus,” Cell,    58:269, 1989.-   Bray, Prasad, Dubay, Hunter, Jeang, Rekosh, Hammarskjold, “A small    element from the Mason-Pfizer monkey virus genome makes human    immunodeficiency virus type 1 expression and replication    Rev-independent,” Proc. Natl. Acad. Sci. 91:1256-60, 1994.-   Brown, Tiley, Cullen, “Efficient polyadenylation within the human    immunodeficiency virus type 1 long terminal repeat requires flanking    U3-specific sequences,” J. Virol., 65:3340-3343, 1991.-   Bulla and Siddiqui, “The hepatitis B virus enhancer modulates    transcription of the hepatitis B virus surface-antigen gene from an    internal location,” J. Virol., 62:1437, 1986.-   Campbell and Villarreal, “Functional analysis of the individual    enhancer core sequences of polyoma virus: cell-specific uncoupling    of DNA replication from transcription,” Mol. Cell. Biol., 8:1993,    1988.-   Campere and Tilghman, “Postnatal repression of the alpha-fetoprotein    gene is enhancer independent,” Genes and Dev., 3:537, 1989.-   Campo, Spandidos, Lang, Wilkie, “Transcriptional control signals in    the genome of bovine papilloma virus type 1,” Nature, 303:77, 1983.-   Carbonelli et al. “A plasmid vector for isolation of strong    promoters in E. coli,” FEMS Microbiol Lett. 177(1):75-82, 1999.-   Case, Price, Jordan, Yu, Wang, Bauer, Haas, Xu, Stripecke, Naldini,    Kohn, Crooks,-   “Stable transduction of quiescent CD34(+)CD38(−) human hematopoietic    cells by HIV-1 based lentiviral vectors,” Proc. Natl. Acad. Sci.    USA, 96:2988-2993, 1999.-   Celander and Haseltine, “Glucocorticoid Regulation of Murine    Leukemia Virus Transcription Elements is Specified by Determinants    Within the Viral Enhancer Region,” J. Virology, 61:269, 1987.-   Celander, Hsu, and Haseltine, “Regulatory Elements Within the Murine    Leukemia Virus Enhancer Regions Mediate Glucocorticoid    Responsiveness,” J. Virology, 62:1314, 1988.-   Chandler, Maler, and Yamamoto, “DNA Sequences Bound Specifically by    Glucocorticoid Receptor in vitro Render a Heterlogous Promoter    Hormone Responsive in vivo,” Cell, 33:489, 1983.-   Chandler et al., “RNA splicing specificity determined by the    coordinated action of RNA recognition motifs in SR proteins,” Proc    Natl Acad Sci USA. 94(8):3596-3601, 1997.-   Chang, Erwin, and Lee, “Glucose-regulated Protein (GRP94 and GRP78)    Genes Share Common Regulatory Domains and are Coordinately Regulated    by Common Trans-acting Factors,” Mol. Cell. Biol., 9:2153, 1989.-   Charneau, Mirambeau, Roux, Paulous, Buc, Clavel, “HIV-1 reverse    transcription: a termination step at the center of the genome,” J.    Mol. Biol. 241:651-662, 1994.-   Chatterjee, Lee, Rentoumis, and Jameson, “Negative Regulation of the    Thyroid-Stimulating Hormone Alpha Gene by Thyroid Hormone: Receptor    Interaction Adjacent to the TATA Box,” Proc Natl. Acad. Sci. U.S.A.,    86:9114, 1989.-   Chen and Okayama, “High-efficiency transformation of mammalian cells    by plasmid DNA,” Mol. Cell. Biol. 7:2745-2752, 1987-   Chemington and Ganem, “Regulation of polyadenylation in human    immunodeficiency virus (HIV): contributions of promoter proximity    and upstream sequences,”Embo. J., 11:1513-1524, 1992.-   Choi, Chen, Kriegler, and Roninson, “An altered pattern of    cross-resistance in multi-drug-resistant human cells results from    spontaneous mutations in the mdr-1 (p-glycoprotein) gene,” Cell,    53:519, 1988.-   Cocea, “Duplication of a region in the multiple cloning site of a    plasmid vector to enhance cloning-mediated addition of restriction    sites to a DNA fragment,”Biotechniques, 23:814-816, 1997.-   Cohen, Walter, and Levinson, “A Repetitive Sequence Element 3′ of    the Human c-Ha-ras1 Gene Has Enhancer Activity,” J. Cell. Physiol.,    5:75, 1987.-   Corbeau, et al., PNAS (U.S.A.) 93(24):14070-14075, 1996.-   Costa, Lai, Grayson, and Daniell, “The Cell-Specific Enhancer of the    Mouse Transthyretin (Prealbumin) Gene Binds a Common Factor at One    Site and a Liver-Specific Factor(s) at Two Other Sites,” Mol. Cell.    Biol., 8:81, 1988.-   Cripe, Haugen, Turk, Tabatabai, Schmid, Durst, Gissmann, Roman, and    Turek, “Transcriptional Regulation of the Human Papilloma Virus-16    E6-E7 Promoter by a Keratinocyte-Dependent Enhancer, and by Viral E2    Trans-Activator and Repressor Gene Products: Implications for    Cervical Carcinogenesis,” EMBO J., 6:3745, 1987.-   Culotta and Hamer, “Fine Mapping of a Mouse Metallothionein Gene    Metal-Response Element,” Mol. Cell. Biol., 9:1376, 1989.-   Dandolo, Blangy, and Kamen, “Regulation of Polyma Virus    Transcription in Murine Embryonal Carcinoma Cells,” J. Virology,    47:55, 1983.-   Das, Klayer, Klasens, van Wamel, “A conserved hairpin motif in the    R-U5 region of the human immunodeficiency virus type 1 RNA genome is    essential for replication,”J. Virol. 71:2346-2356.-   Dao, Hannum, Kohn, Nolta, “FLT3 ligand preserves the ability of    human CD34+ progenitors to sustain long-term hematopoiesis in    immune-deficient mice after ex vivo retroviral-mediated    transduction,” Blood, 89:446-456, 1997.-   Dao, Hashino, Kato, Nolta, “Adhesion to fibronectin maintains    regenerative capacity during ex vivo, culture and transduction of    human hematopoietic stem and progenitor cells,” Blood, 92:4612-4621,    1998.-   Deschamps, Meijlink, and Verma, “Identification of a Transcriptional    Enhancer Element Upstream From the Proto-Oncogene Fos,” Science,    230:1174, 1985.-   De Villiers, Schaffner, Tyndall, Lupton, and Kamen, “Polyoma Virus    DNA Replication Requires an Enhancer,” Nature, 312:242, 1984.-   DeZazzo, Kilpatrick, Imperiale, “Involvement of long terminal repeat    U3 sequences overlapping the transcription control region in human    immunodeficiency virus type 1 mRNA 3′ end formation,” Mol. Cell.    Biol., 11:1624-1630, 1991.-   Donello, Loeb, Hope, “Woodchuck hepatitis virus contains a    tripartite posttranscriptional regulatory element,” J. Virol.,    72:5085-5092, 1998.-   Dorrell, Gan, Pereira, Hawley, Dick, “Expansion of human cord blood    CD34(+)CD38(−) cells in ex vivo culture during retroviral    transduction without a corresponding increase in SCID repopulating    cell (SRC) frequency: dissociation of SRC phenotype and function,”    Blood, 95:102-110, 2000.-   Dull, Zufferey, Kelly, Mandel, Nguyen, Trono, Naldini, “A third    generation lentivirus vector with a conditional packaging    system,” J. Virol., 72:8463-8471, 1998.-   Edbrooke, Burt, Cheshire, and Woo, “Identification of cis-acting    sequences responsible for phorbol ester induction of human serum    amyloid a gene expression via a nuclear-factor-kappa β-like    transcription factor,” Mol. Cell. Biol., 9:1908, 1989.-   Edlund, Walker, Barr, and Rutter, “Cell-specific expression of the    rat insulin gene: evidence for role of two distinct 5′ flanking    elements,” Science, 230:912, 1985.-   Fechheimer, Boylan, Parker, Sisken, Patel and Zimmer, “Transfection    of mammalian cells with plasmid DNA by scrape loading and sonication    loading,” Proc Nat'l. Acad. Sci. USA 84:8463-8467, 1987-   Feng and Holland, “HIV-I Tat Trans-Activation Requires the Loop    Sequence Within Tar,” Nature, 334:6178, 1988.-   Firak and Subramanian, “Minimal Transcription Enhancer of Simian    Virus 40 is a 74-Base-Pair Sequence that Has Interacting Domains,”    Mol. Cell. Biol., 6:3667, 1986.-   Foecking and Hofstetter, “Powerful and Versatile Enhancer-Promoter    Unit for Mammalian Expression Vectors,” Gene, 45(1):101-105, 1986.-   Froehler, Ng, Matteucci, “Synthesis of DNA via deoxynucleoside    H-phosphonate intermediates.” Nuc. Acids Res. 14:5399-407, 1986.-   Fujita, Shibuya, Hotta, Yamanishi, and Taniguchi, “Interferon-Beta    Gene Regulation: Tandemly Repeated Sequences of a Synthetic 6-bp    Oligomer Function as a Virus-Inducible Enhancer,” Cell, 49:357,    1987.-   Gilles, Morris, Oi, and Tonegawa, “A tissue-specific transcription    enhancer element is located in the major intron of a rearranged    immunoglobulin heavy-chain gene,” Cell, 33:717, 1983.-   Gilmartin, Fleming, Oetjen, “Activation of HIV-1 pre-mRNA 3′    processing in vitro requires both an upstream element and TAR,”    Embo. J., 11:4419-4428, 1992.-   Gloss, Bernard, Seedorf, and Klock, “The Upstream Regulatory Region    of the Human Papilloma Virus-16 Contains an E2 Protein-Independent    Enhancer Which is Specific for Cervical Carcinoma Cells and    Regulated by Glucocorticoid Hormones,” EMBO J., 6:3735, 1987.-   Godbout, Ingram, and Tilghman, “Fine-Structure Mapping of the Three    Mouse Alpha-Fetoprotein Gene Enhancers,” Mol. Cell. Biol., 8:1169,    1988.-   Goodbourn, Burstein, and Maniatis, “The Human Beta-Interferon Gene    Enhancer is Under Negative Control,” Cell, 45:601, 1986.-   Goodbourn and Maniatis, “Overlapping Positive and Negative    Regulatory Domains of the Human 13-Interferon Gene,” Proc. Natl.    Acad. Sci. USA, 85:1447, 1988.-   Gopal, “Gene transfer method for transient gene expression, stable    transformation, and cotransformation of suspension cell cultures,”    Mol. Cell. Biol. 5:1188-1190, 1985.-   Gossen and Bujard, Proc. Natl. Acad. Sci., 89:5547-5551, 1992.-   Graham and Van Der Eb, “A new technique for the assay of infectivity    of human adenovirus 5 DNA,” Virology 52:456-467, 1973-   Greco and Dachs, “Gene directed enzyme/prodrug therapy of cancer:    historical appraisal and future prospectives,” J. Cell. Phys. 187:    22-36, 2001-   Greene, Bohnlein, and Ballard, “HIV-1, and Normal T-Cell Growth:    Transcriptional Strategies and Surprises,” Immunology Today, 10:272,    1989-   Grosschedl and Baltimore, “Cell-Type Specificity of Immunoglobulin    Gene Expression is Regulated by at Least Three DNA Sequence    Elements,” Cell, 41:885, 1985.-   Haslinger and Karin, “Upstream Promoter Element of the Human    Metallothionein-II Gene Can Act Like an Enhancer Element,” Proc    Natl. Acad. Sci. U.S.A., 82:8572, 1985.-   Hauber and Cullen, “Mutational Analysis of the    Trans-Activiation-Responsive Region of the Human Immunodeficiency    Virus Type I Long Terminal Repeat,” J. Virology, 62:673, 1988.-   Hen, Borrelli, Fromental, Sassone-Corsi, and Chambon, “A Mutated    Polyoma Virus Enhancer Which is Active in Undifferentiated Embryonal    Carcinoma Cells is not Repressed by Adenovirus-2 E1A Products,”    Nature, 321:249, 1986.-   Hensel, Meichle, Pfizenmaier, and Kronke, “PMA-Responsive 5′    Flanking Sequences of the Human TNF Gene,” Lymphokine Res., 8:347,    1989.-   Herr and Clarke, “The SV40 Enhancer is Composed of Multiple    Functional Elements That Can Compensate for One Another,” Cell,    45:461, 1986.-   Hirochika, Browker, and Chow, “Enhancers and Trans-Acting E2    Transcriptional Factors of Papilloma Viruses,” J. Virol., 61:2599,    1987.-   Hirsch, Gaugler, Deagostini-Bauzin, Bally-Cuif, and Gordis,    “Identification of Positive and Negative Regulatory Elements    Governing Cell-Type-Specific Expression of the    Neural-Cell-Adhesion-Molecule Gene,” Mol. Cell. Biol., 10:1959,    1990.-   Holbrook, Gulino, and Ruscetti, “cis-Acting Transcriptional    Regulatory Sequences in the Gibbon Ape Leukemia Virus (GALV) Long    Terminal Repeat,” Virology, 157:211, 1987.-   Horlick and Benfield, “The upstream muscle-specific enhancer of the    rat muscle creatine kinase gene is composed of multiple elements,”    Mol. Cell. Biol., 9:2396, 1989.-   Huang, Ostrowski, Berard, and Hagar, “Glucocorticoid regulation of    the ha-musv p21 gene conferred by sequences from mouse mammary tumor    virus,” Cell, 27:245, 1981.-   Hug, Costas, Staeheli, Aebi, and Weissmann, “Organization of the    Murine Mx Gene and Characterization of its Interferon- and    Virus-Inducible Promoter,” Mol. Cell. Biol., 8:3065, 1988.-   Hwang, Lim, and Chae, “Characterization of the S-Phase-Specific    Transcription Regulatory Elements in a DNA-Replication-Independent    Testis-Specific H2B (TH2B) Histone Gene,” Mol. Cell. Biol., 10:585,    1990.-   Imagawa, Chiu, and Karin, “Transcription Factor AP-2 Mediates    Induction by Two Different Signal-Transduction Pathways: Protein    Kinase C and cAMP,” Cell, 51:251, 1987.-   Imbra and Karin, “Phorbol Ester Induces the Transcriptional    Stimulatory Activity of the SV40 Enhancer,” Nature, 323:555, 1986.-   Imler, Lemaire, Wasvlyk, and Waslyk, “Negative Regulation    Contributes to Tissue Specificity of the Immunoglobulin Heavy-Chain    Enhancer,” Mol. Cell. Biol, 7:2558, 1987.-   Imperiale and Nevins, “Adenovirus 5 E2 Transcription Unit: an    E1A-Inducible Promoter with an Essential Element that Functions    Independently of Position or Orientation,” Mol. Cell. Biol., 4:875,    1984.-   Jakobovits, Smith, Jakobovits, and Capon, “A Discrete Element 3′ of    Human Immunodeficiency Virus 1 (HIV-1) and HIV-2 mRNA Initiation    Sites Mediates Transcriptional Activation by an HIV    Trans-Activator,” Mol. Cell. Biol., 8:2555, 1988.-   Jameel and Siddiqui, “The Human Hepatitis B Virus Enhancer Requires    Transacting Cellular Factor(s) for Activity,” Mol. Cell. Biol.,    6:710, 1986.-   Jaynes, Johnson, Buskin, Gartside, and Hauschka, “The Muscle    Creatine Kinase Gene is Regulated by Multiple Upstream Elements,    Including a Muscle-Specific Enhancer,” Mol. Cell. Biol., 8:62, 1988.-   Johnson, Wold, and Hauschka, “Muscle creatine kinase sequence    elements regulating skeletal and cardiac muscle expression in    transgenic mice,” Mol. Cell. Biol., 9:3393, 1989.-   Kadesch and Berg, “Effects of the Position of the Simian Virus 40    Enhancer on Expression of Multiple Transcription Units in a Single    Plasmid,” Mol. Cell. Biol., 6:2593, 1986.-   Kafri, et al., “Sustained expression of genes delivered directly    into liver and muscle by lentiviral vectors,” Nat. Genetics,    17:314-317, 1997.-   Karin, Haslinger, Heguy, Dietlin, and Cooke, “Metal-Responsive    Elements Act as Positive Modulators of Human Metallothionein-IIA    Enhancer Activity,” Mol. Cell. Biol., 7:606, 1987.-   Katinka, Yaniv, Vasseur, and Blangy, “Expression of Polyoma Early    Functions in Mouse Embryonal Carcinoma Cells Depends on Sequence    Rearrangements in the Beginning of the Late Region,” Cell, 20:393,    1980.-   Kawamoto, Making, Niw, Sugiyama, Kimura, Anemura, Nakata, and    Kakunaga, “Identification of the Human Beta-Actin Enhancer and its    Binding Factor,” Mol. Cell. Biol., 8:267, 1988.-   Kiledjian, Su, Kadesch, “Identification and characterization of two    functional domains within the murine heavy-chain enhancer,” Mol.    Cell. Biol., 8:145, 1988.-   Klages, Zufferey, Trono, “A stable system for the high-titer    production of multiply aattenuated lentiviral vectors,” Mol. Ther.    2:170-176, 2000.-   Klamut, Gangopadyhay, Worton, and Ray, “Molecular and Functional    Analysis of the Muscle-Specific Promoter Region of the Duchenne    Muscular Dystrophy Gene,”Mol. Cell. Biol., 10:193, 1990.-   Klein et al., “High-velocity microprojectiles for delivering nucleic    acids into living cells,” Nature, 327:70-73, 1987.-   Koch, Benoist, and Mathis, “Anatomy of a new β-cell-specific    enhancer,” Mol. Cell. Biol., 9:303, 1989.-   Kohn, Nolta, Weinthal, Balmer, Yu, Lilley, Crooks, “Toward gene    therapy for Gaucher disease,” Hum. Gene Ther., 2:101-105, 1991.-   Kotsopoulou, Kim, Kingsman, Kingsman, Mitrophanous. “A    Rev-independent human immunodeficiency virus type 1 (HIV-1)-based    vector that exploits a codon-optimized HIV-1 gag-pol gene,” J.    Virol. 74:4839-52, 2000.-   Kraus et al., “Alternative promoter usage and tissue specific    expression of the mouse somatostatin receptor 2 gene,” FEBS Lett.,    428(3):165-170, 1998.-   Kriegler and Botchan, “A retrovirus LTR contains a new type of    eukaryotic regulatory element,” In: Eukaryotic Viral Vectors,    Gluzman (ed.), Cold Spring Harbor, Cold Spring Harbor Laboratory,    NY, 1982.-   Kriegler et al., “Promoter substitution and enhancer augmentation    increases the penetrance of the sv40 a gene to levels comparable to    that of the harvey murine sarcoma virus ras gene in morphologic    transformation,” In: Gene Expression, Alan Liss (Ed.), Hamer and    Rosenberg, New York, 1983.-   Kriegler et al., “Viral Integration and Early Gene Expression Both    Affect the Efficiency of SV40 Transformation of Murine Cells:    Biochemical and Biological Characterization of an SV40 Retrovirus,”    In: Cancer Cells 2/Oncogenes and Viral Genes, Van de Woude et al.    (eds), Cold Spring Harbor, Cold Spring Harbor Laboratory, 1984.-   Kriegler, Perez, Defay, Albert and Liu, “A Novel Form of    TNF/Cachectin Is a Cell-Surface Cytotoxix Transmembrane Protein:    Ramifications for the Complex Physiology of TNF,” Cell, 53:45, 1988.-   Kuhl, De La Fuenta, Chaturvedi, Parinool, Ryals, Meyer, and    Weissman, “Reversible Silencing of Enhancers by Sequences Derived    From the Human IFN-alpha Promoter,” Cell, 50:1057, 1987.-   Kunz, Zimmerman, Heisig, and Heinrich, “Identification of the    Promoter Sequences Involved in the Interleukin-6-Dependent    Expression of the Rat Alpha-2-Macroglobulin Gene,” Nucl. Acids Res.,    17:1121, 1989.-   Lareyre et al., “A 5-kilobase pair promoter fragment of the murine    epididymal retinoic acid-binding protein gene drives the    tissue-specific, cell-specific, and androgen-regulated expression of    a foreign gene in the epididymis of transgenic mice,” J Biol. Chem.,    274(12):8282-8290, 1999.-   Larsen, Harney, and Moore, “Repression medaites cell-type-specific    expression of the rat growth hormone gene,” Proc Natl. Acad. Sci.    USA., 83:8283, 1986.-   Laspia, Rice, and Mathews, “HIV-1 Tat protein increases    transcriptional initiation and stabilizes elongation,” Cell, 59:283,    1989.-   Latimer, Berger, and Baumann, “Highly conserved upstream regions of    the alpha.sub.1-antitrypsin gene in two mouse species govern    liver-specific expression by different mechanisms,” Mol. Cell.    Biol., 10:760, 1990.-   Lee, Mulligan, Berg, and Ringold, “Glucocorticoids Regulate    Expression of Dihydrofolate Reductase cDNA in Mouse Mammary Tumor    Virus Chimaeric Plasmids,” Nature, 294:228, 1981.-   Lee et al., “Activation of beta3-adrenoceptors by exogenous dopamine    to lower glucose uptake into rat adipocytes,” J Auton Nery Syst.    74(2-3):86-90, 1997.-   Levenson et al., “Internal ribosomal entry site-containing    retroviral vectors with green fluorescent protein and drug    resistance markers,” Human Gene Therapy, 9:1233-1236, 1998.-   Levinson, Khoury, VanDeWoude, and Gruss, “Activation of SV40 Genome    by 72-Base-Pair Tandem Repeats of Moloney Sarcoma Virus,” Nature,    295:79, 1982. Lewis and Emerman, “Passage through mitosis is    required for oncoretroviruses but not for the human immunodeficiency    virus,” J. Virol., 68:510-516, 1994.-   Lin, Cross, Halden, Dragos, Toledano, and Leonard, “Delineation of    an enhancerlike positive regulatory element in the interleukin-2    receptor .alpha.-chain gene,” Mol. Cell. Biol., 10:850, 1990.-   Luria, Gross, Horowitz, and Givol, “Promoter Enhancer Elements in    the Rearranged Alpha-Chain Gene of the Human T-Cell Receptor,” EMBO    J., 6:3307, 1987.-   Lusky, Berg, Weiher, and Botchan, “Bovine Papilloma Virus Contains    an Activator of Gene Expression at the Distal End of the Early    Transcription Unit,” Mol. Cell. Biol. 3:1108, 1983.-   Lusky and Botchan, “Transient Replication of Bovine Papilloma Virus    Type 1 Plasmids: cis and trans Requirements,” Proc Natl. Acad. Sci.    U.S.A., 83:3609, 1986.-   Majors and Varmus, “A Small Region of the Mouse Mammary Tumor Virus    Long Terminal Repeat Confers Glucocorticoid Hormone Regulation on a    Linked Heterologous Gene,” Proc. Natl. Acad. Sci. U.S.A., 80:5866,    1983.-   Marthas et al. J. Virol., 67:6047-6055, 1993.-   Mazurier, Moreau-Gaudry, Maguer-Satta, Salesse, Pigeonnier-Lagarde,    Ged, Belloc, Lacombe, Mahon, Reiffers, de Verneuil, “Rapid analysis    and efficient selection of human transduced primitive hematopoietic    cells using the humanized S65T green fluorescent protein,” Gene    Ther., 5:556-562, 1998.-   McNeall, Sanchez, Gray, Chesterman, and Sleigh, “Hyperinducible Gene    Expression From a Metallotionein Promoter Containing Additional    Metal-Responsive Elements,” Gene, 76:81, 1989.-   Miksicek, Heber, Schmid, Danesch, Posseckert, Beato, and Schutz,    “Glucocorticoid Responsiveness of the Transcriptional Enhancer of    Moloney Murine Sarcoma Virus,” Cell, 46:203, 1986.-   Miyoshi, Smith, Mosier, Verma, Torbett, “Transduction of human CD34+    cells that mediate long-term engraftment of NOD/SCID mice by HIV    vectors,” Science, 283:682-686, 1999.-   Mizushima and Nagata, “pEF-BOS, a powerful mammalian expression    vector,” Nucleic Acids Res., 18:5322, 1990.-   Mordacq and Linzer, “Co-localization of Elements Required for    Phorbol Ester Stimulation and Glucocorticoid Repression of    Proliferin Gene. Expression,” Genes and Dev., 3:760, 1989.-   Moreau, Hen, Wasylyk, Everett, Gaub, and Chambon, “The SV40    base-repair repeat has a striking effect on gene expression both in    sv40 and other chimeric recombinants,” Nucl. Acids Res., 9:6047,    1981.-   Muesing et al., Cell, 48:691, 1987.-   Naldini, Blomer, gallay, Ory, Mulligan, Gage, Verma, Trono, “In vivo    gene delivery and stable transduction of nondividing cells by a    lentiviral vector,” Science, 272:263-267, 1996a.-   Naldini, Blomer, Gage, Trono, Verma, “Efficient transfer,    integration, and sustained long-term expression of the transgene in    adult rat brains injected with a lentiviral vector,” Proc. Natl.    Acad. Sci. USA, 93:11382-11388, 1996b.-   Naldini, “Lentiviruses as gene transfer agents for delivery to    non-dividing cells,” Curr. Opin. Biotechnol. 9:457-463, 1998.-   Ng, Gunning, Liu, Leavitt, and Kedes, “Regulation of the Human    Beta-Actin Promoter by Upstream and Intron Domains,” Nuc. Acids    Res., 17:601, 1989.-   Nomoto et al., “Cloning and characterization of the alternative    promoter regions of the human LIMK2 gene responsible for alternative    transcripts with tissue-specific expression,” Gene, 236(2):259-271,    1999.-   Omitz, Hammer, Davison, Brinster, and Palmiter, “Promoter and    enhancer elements from the rat elastase i gene function    independently of each other and of heterologous enhancers,” Mol.    Cell. Biol. 7:3466, 1987.-   Ondek, Sheppard, and Herr, “Discrete Elements Within the SV40    Enhancer Region Display Different Cell-Specific Enhancer    Activities,” EMBO J., 6:1017, 1987.-   Ory et al., Proc. Natl. Acad. Sci., 93:11400-11406, 1996.-   Palmiter, Chen, and Brinster, “Differential regulation of    metallothionein-thymidine kinase fusion genes in transgenic mice and    their offspring,” Cell, 29:701, 1982.-   Pech, Rao, Robbins, and Aaronson, “Functional identification of    regulatory elements within the promoter region of platelet-derived    growth factor 2,” Mol. Cell. Biol., 9:396, 1989.-   Perez-Stable and Constantini, “Roles of fetal γ-globin promoter    elements and the adult β-globin 3′ enhancer in the stage-specific    expression of globin genes,” Mol. Cell. Biol., 10:1116, 1990.-   Piacibello, Sanavio, Severino, Dane, Gammaitoni, Fagioli,    Perissinotto, Cavalloni, Kollet Lapidot, Aglietta, “Engraftment in    nonobese diabetic severe combined immunodeficient mice of human    CD34(+) cord blood cells after ex vivo expansion: evidence for the    amplification and self-renewal of repopulating stem cells,” Blood,    93:3736-3749, 1999.-   Picard and Schaffner, “A Lymphocyte-Specific Enhancer in the Mouse    Immunoglobulin Kappa Gene,” Nature, 307:83, 1984.-   Pinkert, Omitz, Brinster, and Palmiter, “An albumin enhancer located    10 kb upstream functions along with its promoter to direct    efficient, liver-specific expression in transgenic mice,” Genes and    Dev., 1:268, 1987.-   Ponta, Kennedy, Skroch, Hynes, and Groner, “Hormonal Response Region    in the Mouse Mammary Tumor Virus Long Terminal Repeat Can Be    Dissociated From the Proviral Promoter and Has Enhancer Properties,”    Proc. Natl. Acad. Sci. U.S.A., 82:1020, 1985.-   Porton, Zaller, Lieberson, and Eckhardt, “Immunoglobulin heavy-chain    enhancer is required to maintain transfected .gamma.2a gene    expression in a pre-b-cell line,”Mol. Cell. Biol., 10:1076, 1990.-   Potter et al., “Enhancer-dependent expression of human k    immunoglobulin genes introduced into mouse pre-B lymphocytes by    electroporation,” Proc Nat'l Acad. Sci. USA, 81:7161-7165, 1984.-   Queen and Baltimore, “Immunoglobulin Gene Transcription is Activated    by Downstream Sequence Elements,” Cell, 35:741, 1983.-   Quinn, Farina, Gardner, Krutzsch, and Levens, “Multiple components    are required for sequence recognition of the ap1 site in the gibbon    ape leukemia virus enhancer,” Mol. Cell. Biol., 9:4713, 1989.-   Redondo, Hata, Brocklehurst, and Krangel, “A T-Cell-Specific    Transcriptional Enhancer Within the Human T-Cell Receptor .delta.    Locus,” Science, 247:1225, 1990.-   Resendez Jr., Wooden, and Lee, “Identification of highly conserved    regulatory domains and protein-binding sites in the promoters of the    rat and human genes encoding the stress-inducible 78-kilodalton    glucose-regulated protein,” Mol. Cell. Biol., 8:4579, 1988.-   Reisman and Rotter, “Induced Expression From the Moloney Murine    Leukemia Virus Long Terminal Repeat During Differentiation of Human    Myeloid Cells is Mediated Through its Transcriptional Enhancer,”    Mol. Cell. Biol., 9:3571, 1989.-   Remington's Pharmaceutical Sciences, 15^(th) Ed., pages 1035-1038    and 1570-1580.-   Ripe, Lorenzen, Brenner, and Breindl, “Regulatory elements in the 5′    flanking region and the first intron contribute to transcriptional    control of the mouse alpha-1-type collagen gene,” Mol. Cell. Biol.,    9:2224, 1989.-   Rippe, Brenner and Leffert, “DNA-mediated gene transfer into adult    rat hepatocytes in primary culture,” Mol. Cell. Biol., 10:689-695,    1990.-   Riffling, Coutinho, Amarm, and Kolbe, “AP-1/jun-binding Sites    Mediate Serum Inducibility of the Human Vimentin Promoter,” Nuc.    Acids Res., 17:1619, 1989.-   Roe, Reynolds, Yu, Brown, “Integration of murine leukemia virus DNA    depends on mitosis,” Embo. J., 12:2099-2108, 1993.-   Rosen, Sodroski, and Haseltine, “The location of cis-acting    regulatory sequences in the human t-cell lymphotropic virus type III    (HTLV-111/LAV) long terminal repeat,”Cell, 41:813, 1988.-   Sakai, Helms, Carlstedt-Duke, Gustafsson, Rottman, and Yamamoto,    “Hormone-Mediated Repression: A Negative Glucocorticoid-Response    Element From the Bovine Prolactin Gene,” Genes and Dev., 2:1144,    1988.-   Sambrook, Fritsch, Maniatis, In: Molecular Cloning: A Laboratory    Manual 2 rev. ed., Cold Spring Harbor, Cold Spring Harbor Laboratory    Press, 1(77):19-17.29, 1989.-   Satake, Furukawa, and Ito, “Biological activities of    oligonucleotides spanning the 19 point mutation within the enhancer    region of polyoma virus DNA,” J. Virology, 62:970, 1988.-   Scharfmann, Axelrod, Verma, “Long-term in vivo expression of    retrovirus-mediated gene transfer in mouse fibroblast implants,”    Proc. Natl. Acad. Sci. USA, 88:4626-4630, 1991.-   Schaffner, Schirm, Muller-Baden, Wever, and Schaffner, “Redundancy    of Information in Enhancers as a Principle of Mammalian    Transcription Control,” J. Mol. Biol., 201:81, 1988.-   Schmid, Uittenbogaart, Keld, Giorgi, “A rapid method for measuring    apoptosis and dual-color immunofluorescence by single laser flow    cytometry,” J. Immunol. Methods, 170:145-157, 1994.-   Searle, Stuart, and Palmiter, “Building a metal-responsive promoter    with synthetic regulatory elements,” Mol. Cell. Biol., 5:1480, 1985.-   Sharp and Marciniak, “HIV Tar: an RNA Enhancer?,” Cell, 59:229,    1989.-   Shaul and Ben-Levy, “Multiple Nuclear Proteins in Liver Cells are    Bound to Hepatitis B Virus Enhancer Element and its Upstream    Sequences,” EMBO J., 6:1913, 1987.-   Sherman, Basta, Moore, Brown, and Ting, “Class II Box Consensus    Sequences in the HLA-DR.alpha. Gene: Transcriptional Function and    Interaction with Nuclear Proteins,” Mol. Cell. Biol., 9:50, 1989.-   Sleigh and Lockett, “SV40 Enhancer Activation During    Retinoic-Acid-Induced Differentiation of F9 Embryonal Carcinoma    Cells,” J. EMBO, 4:3831, 1985.-   Spalholz, Yang, and Howley, “Transactivation of a Bovine Papilloma    Virus Transcriptional Regulatory Element by the E2 Gene Product,”    Cell, 42:183, 1985.-   Spandau and Lee, “Trans-Activation of Viral Enhancers by the    Hepatitis B Virus X Protein,” J. Virology, 62:427, 1988.-   Spandidos and Wilkie, “Host-Specificities of Papilloma Virus,    Moloney Murine Sarcoma Virus and Simian Virus 40 Enhancer    Sequences,” EMBO J., 2:1193, 1983.-   Stephens and Hentschel, “The Bovine Papilloma Virus Genome and its    Uses as a Eukaryotic Vector,” Biochem. J., 248:1, 1987.-   Stuart, Searle, and Palmiter, “Identification of Multiple Metal    Regulatory Elements in Mouse Metallothionein-I Promoter by Assaying    Synthetic Sequences,” Nature, 317:828, 1985.-   Sullivan and Peterlin, “Transcriptional Enhancers in the HLA-DQ    Subregion,” Mol. Cell. Biol., 7:3315, 1987.-   Sutton, Reitsma, Uchida, Brown, “Transduction of human progenitor    hematopoietic stem cells by human immunodeficiency virus type    1-based vectors is cell cycle dependent,” J. Virol., 73:3649-3660,    1999.-   Sutton, Wu, Rigg, Bohnlein, Brown, “Human immunodeficiency virus    type 1 vectors efficiently transduce human hematopoietic stem    cells,” “J. Virol., 72:5781-5788, 1998.-   Swartzendruber and Lehman, “Neoplastic Differentiation: Interaction    of Simian Virus 40 and Polyoma Virus with Murine Teratocarcinoma    Cells,” J. Cell. Physiology, 85:179, 1975.-   Takebe, Seiki, Fujisawa, Hoy, Yokota, Arai, Yoshida, and Arai,    “SR.alpha. Promoter: An-   Efficient and Versatile Mammalian cDNA Expression System Composed of    the Simian Virus 40 Early Promoter and the R-U5 Segment of Human    T-Cell. Leukemia Virus Type 1 Long Terminal Repeat,” Mol. Cell.    Biol., 8:466, 1988.-   Taylor and Kingston, “E1A Trans-Activation of Human HSP70 Gene    Promoter Substitution Mutants is Independent of the Composition of    Upstream and TATA Elements,” Mol. Cell. Biol., 10:176, 1990.-   Taylor and Kingston, “Factor Substitution in a Human HSP70 Gene    Promoter: TATA-Dependent and TATA-Independent Interactions,” Mol.    Cell. Biol., 10:165, 1990.-   Taylor, Solomon, Weiner, Paucha, Bradley, and Kingston, “Stimulation    of the Human Heat-Shock Protein 70 Promoter in vitro by Simian Virus    40 Large T Antigen,” J. Biol. Chem., 264:15160, 1989.-   Tavernier, Gheysen, Duerinck, Can Der Heyden, and Fiers, “Deletion    Mapping of the Inducible Promoter of Human IFN-beta Gene,” Nature,    301:634, 1983.-   Thiesen, Bosze, Henry, and Charnay, “A DNA Element Responsible for    the Different Tissue Specificities of Friend and Moloney Retroviral    Enhancers,” J. Virology, 62:614, 1988.-   Tronche, Rollier, Bach, Weiss, and Yaniv, “The Rat Albumin Promoter:    Cooperation with Upstream Elements is Required When Binding of    APF/HNF 1 to the Proximal Element is Partially Impaired by Mutation    or Bacterial Methylation,”Mol. Cell. Biol., 9:4759, 1989.-   Tronche, Rollier, Herbomel, Bach, Cereghini, Weiss, and Yaniv,    “Anatomy of the Rat Albumin Promoter,” Mol. Biol. Med., 7:173, 1990.-   Trudel and Constantini, “A 3′ Enhancer Contributes to the    Stage-Specific Expression of the human Beta-Globin Gene,” Genes and    Dev., 6:954, 1987.-   Tsumaki et al., “Modular arrangement of cartilage- and neural    tissue-specific cis-elements in the mouse alpha2(XI) collagen    promoter,” J Biol. Chem. 273(36):22861-22864, 1998.-   Tur-Kaspa, Teicher, Levine, Skoultchi and Shafritz, “Use of    electroporation to introduce biologically active foreign genes into    primary rat hepatocytes,” Mol. Cell. Biol., 6:716-718, 1986.-   Tyndall, La Mantia, Thacker, Favaloro, and Kamen, “A Region of the    Polyoma Virus Genome Between the Replication Origin and Late    Protein-Coding Sequences is Required in cis for Both Early Gene    Expression and Viral DNA Replication,”Nuc. Acids. Res., 9:6231,    1981.-   Uchida, Sutton, Friera, He, Reitsma, Chang, Veres, Scollay,    Weissman, “HIV, but not murine leukemia virus, vectors mediate high    efficiency gene transfer into freshly isolated G0/G1 human    hematopoietic stem cells,” Proc. Natl. Acad. Sci. USA,    95:11939-11944, 1998.-   Ueda, Tsuji, Yoshino, Ebihara, Yagasaki, Hisakawa, Mitsui, Manabe,    Tanaka, Kobayashi, Ito, Yasukawa, Nakahata, “Expansion of human    NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand,    thrombopoietin, IL-6, and soluble IL-6 receptor,” J. Clin. Invest.,    105:1013-1021, 2000.-   Unutmaz, Kewal, Ramani, Marmon, Littman, “Cytokine signals are    sufficient for HIV-1 infection of resting human T lymphocytes,” J.    Exp. Med., 189:1735-1746, 1999.-   Valsamakis, Schek, Alwine, “Elements upstream of the AAUAAA within    the human immunodeficiency virus polyadenylation signal are required    for efficient polyadenylation in vitro,” Mol. Cell. Biol.,    12:3699-3705, 1992.-   Valsamakis, Zeichner, Carswell, Alwine, “The human immunodeficiency    virus type 1 polyadenylylation signal: a 3′ long terminal repeat    element upstream of the AAUAAA necessary for efficient    polyadenylylation,” Proc. Natl. Acad. Sci. USA, 88:2108-2112, 1991.-   Vannice and Levinson, “Properties of the Human Hepatitis B Virus    Enhancer: Position Effects and Cell-Type Nonspecificity,” J.    Virology, 62:1305, 1988.-   Vasseur, Kress, Montreau, and Blangy, “Isolation and    Characterization of Polyoma Virus Mutants Able to Develop in    Multipotential Murine Embryonal Carcinoma Cells,”Proc Natl. Acad.    Sci. U.S.A., 77:1068, 1980.-   Wang and Calame, “SV40 enhancer-binding factors are required at the    establishment but not the maintenance step of enhancer-dependent    transcriptional activation,” Cell, 47:241, 1986.-   Watanabe et al., “Gene transfection of mouse primordial germ cells    in vitro and analysis of their survival and growth control,    Experimental Cell Research, 230:76-83, 1997.-   Weber, De Villiers, and Schaffner, “An SV40 ‘Enhancer Trap’    Incorporates Exogenous Enhancers or Generates Enhancers From its Own    Sequences,” Cell, 36:983, 1984.-   Weinberger, Jat, and Sharp, “Localization of a Repressive Sequence    Contributing to B-cell Specificity in the Immunoglobulin Heavy-Chain    Enhancer,” Mol. Cell. Biol., 8:988, 1984.-   Winoto and Baltimore, “αβ-lineage-specific Expression of the α    T-Cell Receptor Gene by Nearby Silencers,” Cell, 59:649, 1989.-   Wu et al., “Promoter-dependent tissue-specific expressive nature of    imprinting gene, insulin-like growth factor II, in human tissues,”    Biochem Biophys Res Commun. 233(1):221-226, 1997.-   Wu, Wakefield, Liu, Xiao, Kralovics, Prchal, Kappes, “Development of    a novel trans-lentiviral vector that affords predictable safety,”    Mol. Ther. 2:47-55, 2000.-   Yang, Burkholder, Roberts, Martinell and McCabe, “In vivo and in    vitro gene transfer to mammalian somatic cells by particle    bombardment,” Proc Nat'l Acad. Sci. USA, 87:9568-9572, 1990.-   Yutzey, Kline, and Konieczny, “An Internal Regulatory Element    Controls Troponin I Gene Expression,” Mol. Cell. Biol., 9:1397,    1989.-   Zennou, Petit, Guetard, Nerhbass, Mantagnier, Charneau, “HIV-1    genome nuclear import is mediated by a central DNA flap,” Cell    101:173-185, 2000.-   Zhao-Emonet et al., “The equine herpes virus 4 thymidine kinase is a    better suicide gene than the human herpes virus 1 thymidine kinase,”    Gene Ther. 6(9):1638-1642, 1999.-   Zufferey, Nagy, Mandel, Naldini, Trono, “Multiply attenuated    lentiviral vector achieves efficient gene delivery in vivo,” Nat.    Biotechnol., 15:871-875, 1997.-   Zufferey, Dull, Mandel, Bukovsky, Quiroz, Naldini, Trono,    “Self-inactivating lentivirus vector for safe and efficient in vivo    gene delivery,” J. Virol., 72:9873-9880, 1998.-   Zufferey, Donello, Trono, Hope, “Woodchuck hepatitis virus    posttranscriptional regulatory element enhances expression of    transgenes delivered by retroviral vectors,” J. Virol.,    73:2886-2892, 1999.-   Zufferey and Trono, Current Protocols in Neuroscience: unit 4.21:    “High-titer production of lentiviral vectors,” John Wiley & Sons,    New York, 2000.

1. A packaging plasmid comprising a stuffer sequence and a cPPT/cTSregion that has reduced replication activity relative to wild-typecPPT/cTS replication activity.
 2. The packaging plasmid of claim 1,wherein the cPPT/cTS region comprises SEQ ID NO:4. 3.-5. (canceled) 6.The packaging plasmid of claim 1, wherein the stuffer sequence is ofsufficient length to effectively provide a lentivirus having at leastthe size of a wild-type lentiviral genome.
 7. The packaging plasmid ofclaim 1, wherein the stuffer sequence encodes a drug sensitivity gene.8. The packaging plasmid of claim 7, wherein the drug sensitivity geneis a thymidine kinase gene.
 9. The packaging plasmid of claim 7, whereinthe drug sensitivity gene is a cytosine deaminase gene.
 10. Thepackaging plasmid of claim 7, wherein the stuffer sequence comprises theIRES-tk cassette.
 11. The packaging plasmid of claim 1, furthercomprising an RRE.
 12. The packaging plasmid of claim 1, furthercomprising a constitutive RNA export element.
 13. The packaging plasmidof claim 11, further comprising a pol gene.
 14. The packaging plasmid ofclaim 13, wherein the stuffer sequence is between the pol gene and theRRE. 15.-103. (canceled)